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J Med Assoc Thai Vol. 100 Suppl. 5 2017 S241
Correspondence to:
Itharat A, Department of Applied Thai Traditional Medicine,
Faculty of Medicine, Thammasat University, Rangsit,
Klongluang, Pathumthani 12120, Thailand.
Phone & Fax: +66-2-9269749
E-mail: iarunporn@yahoo.com
J Med Assoc Thai 2017; 100 (Suppl. 5): S241-S249
Full text. e-Journal: http://www.jmatonline.com
In Vitro Free Radical Scavenging and Cell-Based
Antioxidant Activities of Kheaw-Hom Remedy Extracts
and Its Plant Ingredients
Kulisara Ouncharoen BMTA*,
Arunporn Itharat PhD**,***, Pannawat Chaiyawatthanananthn PhD**,***
* Student of Master of Science in Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University,
Pathumthani, Thailand
** Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Klongluang,
Pathumthani, Thailand
*** Center of Excellence on Applied Thai Traditional Medicine Research (CEATMR), Faculty of Medicine,
Thammasat University, Klongluang, Pathumthani, Thailand
Background: The oxidative stress (OS) and antioxidants play a key role in the pathogenesis of inflammatory diseases such
as fever which is promoted by the production of reactive oxygen species and impaired antioxidant defense mechanisms. The
Kheaw-Hom remedy is popularly used as anti-pyretic drug in Thai traditional medicine.
Objective: To investigate antioxidant activity of the ethanolic and aqueous extracts of Kheaw-Hom remedy and its plant
ingredients by three assays such as DPPH, ABTS radical scavenging assays and NBT dye reduction assay.
Material and Method: The extract procedures were maceration method with 95% ethanol, dried by an evaporator, or the
decoction by boiling in water, filtrated, dried by lyophilizer. In the preliminary studies, all extracts were evaluated for
antioxidant activity through two chemical assays: DPPH radical-scavenging and ABTS radical-scavenging assay, as well as
through cell-based assay: scavenging capacity of intracellular ROS in HL-60 cells using the NBT reduction.
Results: The ethanolic extract of Khaew-Hom remedy showed higher antioxidant activity using DPPH and ABTS assays but
it had no antioxidant activity using cell-based assay (EC50 = 16.96, 30.91 and IC50 >100
μ
g/mL, respectively). The ethanolic
extract of Cyathea gigantea and Tacca chantrieri showed the highest antioxidant activity using DPPH assay with EC50 = 7.55
and 8.00
μ
g/mL, respectively. The ethanolic extract of Dracaena loureiri and Globba malaccensis exhibited the best antioxidant
activity using ABTS radical scavenging with EC50 = 7.88 and 8.06
μ
g/mL, respectively. For the NBT dye reduction assay, only
the ethanolic and aqueous extracts of Tacca chantrieri were effective having IC50 = 63.38 and 70.65
μ
g/mL, respectively.
Conclusion: The ethanolic of Khaew-Hom showed antioxidant activity only with chemical based assay but both ethanolic and
aqueous extracts of Tacca chantrieri (rhizome) showed high antioxidant activities on chemical-based and cell line-based
assay. Thus, this plant should be developed to be health products in the future.
Keywords: Antioxidant activities, Kheaw-Hom, DPPH, ABTS, NBT assay, HL-60
Free radicals are products from the metabolism
of various substances in the liver. The stimuli of free
radicals production are pollution, dust, cigarette smoke,
unsaturated fat food, sunlight, heat, chemicals and
medicine(1). They can stimulate immune system to
produce radical for eliminate the disguise. The
downside of free radicals are, if there are too many free
radicals, they destroy tissue proteins and cells,
including white blood cells(2). On the opposite side,
benefit of free radicals is killing of antigens such as
bacteria, virus or parasite and it implicate in
inflammation(3,4). Inflammation is immune exhibition and
appears in many diseases such as fever, recurrent
aphthous stomatitis(5). For example, exanthematous
fever, the cause of this fever could be viral, bacterial
infection and allergic medicine(6). The inflammatory
condition can cause progessive disease. There is
support evidence that ROS are essential second
messengers in innate and adaptive immune cells and
excessive of ROS within immune cells can result in
S242 J Med Assoc Thai Vol. 100 Suppl. 5 2017
hyperactivation of inflammatory responses(7). So that,
if ROS decreased, the inflammation would be reduced.
In Thai traditional medicine, there are many
antipyretics, one of them is Kheaw-Hom remedy. It is
antipyretic for exanthematous fever in Thai traditional
medicine. It has been published in National List of
Essential Herbal Medicines, Ministry of Public Health,
2013(8). This remedy consists of eighteen Thai herbs
shown in Table 1 with each herb in equal amount. Some
herbs in remedy have been previously studied for
antioxidant activity such as Mimusops elengi Linn(9),
Mesua ferrea Linn(10), Mammea siamensis Kosterm(11),
Nelumbo nucifera Gaertn(12), Sophora exigua Craib(13),
Vetiveria zizanioides (L.) Nash ex Small(14), but
Kheaw-Hom remedy has not been studied. So that, the
objective of this study was to investigate the
antioxidant activity of the ethanolic and aqueous
extracts of Kheaw-Hom remedy and its plant
ingredients.
Material and Method
Chemicals and reagents
95% ethanol (CMJ Anchor company,
Thailand), Distilled water (Milford, USA), 2, 2-diphenyl-
l-picrylhydrazyl (Fluka, Germany), Butylated
hydroxytoluene (BHT) (Fluka, Germany), 2, 2’-azinobis-
(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma-
ALDRICH, USA), 6-hydroxy-2, 5, 7, 8-tetramethyl
chroman-2-carboxylic acid (Trolox) (Sigma-ALDRICH,
USA), Potassium sulfate (Sigma-ALDRICH, USA),
Dimethyl sulfoxide (CH3)2 SO (DMSO) (RCI Labscan,
Thailand), Nitroblue tetrazolium chloride (NBT) (Sigma,
USA), Phorbol myristate acetate (PMA) (Sigma, USA),
Thiazolyl blue tetrazolium bromide (MTT) (Sigma,
USA).
Cell lines and culture conditions
Human leukemia cell line HL-60 cell was
purchased from ATCC. The cells were maintained by
twice weekly passages in RPMI-1640 medium
supplemented with 10% FBS, 1% Penicillin-
Streptomycins and incubated at 37°C in a 5% CO2.
Plant materials
The parts of 18 plants in Kheaw-Hom remedy
were collected from several regions of Thailand in 2015,
with voucher specimen numbers shown in Table 1.
The voucher specimens were carried out at the
herbarium of Southern Center of Thai Medicinal Plants
at Faculty of Pharmaceutical Sciences, Prince of Songkla
University, Songkhla, Thailand.
Preparation of crude extracts
All plants were washed, sliced to small pieces,
dried in an oven at temperature 50°C and ground to
powder and extracted by maceration with 95% ethanol
and boiling in water as ethanolic and aqueous extract,
respectively. The ethanolic extract was prepared by
maceration of Kheaw-Hom remedy (1,170 grams), and
where each of its herbal ingredients (65 grams of each)
was macerated with 95% ethanol for 3 days. Filtrate
was obtained using Whatman No. 1 filter paper and
concentrated to dryness by an evaporator (Rotavapor
R-205, Germany). The aqueous extract of Kheaw-Hom
remedy (1,170 grams) and its herbal ingredients (65
grams of each) were prepared by boiling in distilled
water. The duration of decoction was 15 min. The
extracts were filtered through a Whatman No. 1 filter
paper and dried by lyophilization. The crude extracts
were kept at -20°C untill used.
Determination of antioxidant activity
DPPH radical scavenging assay(15)
The antioxidant activity was determined
using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Sample
was dissolved in absolute ethanol or distilled water in
various concentrations including 100, 50, 10 and 1 μg/
mL. 100 μL of samples were transferred into a 96-well
microplate. Then 100 μl of 6 x 10-5 M DPPH (in absolute
ethanol) was added into each well. After incubation for
30 min in the dark at room temperature, the absorbance
was measured at 520 nm, where BHT was used as a
positive control. The concentration of antioxidant
needed to decrease the initial DPPH concentration
(EC50) by 50% is a parameter widely used to measure
the antioxidant activity. The scavenging activity was
calculated as percentage inhibition in the formulae
below:
% Inhibition = ((Mean of ODControl - Mean of ODsample)/
Mean of ODControl) x 100
Effective concentration of sample required to
scavenge DPPH radical by 50% (EC50) was obtained
by linear regression analysis of the dose-response
curve of % inhibition versus concentration, and EC50 is
calculated using prism program. All determinations were
carried out in triplicate.
ABTS radical scavenging assay(16)
The antioxidant activity was determined 2.45
mM ABTS•+ solution was prepared using potassium
persulfate diluted with DI water to get the absorbance
J Med Assoc Thai Vol. 100 Suppl. 5 2017 S243
Scientific Name Family Name Thai Name Part used Flavor Voucher number Ratio Thai traditional used
(%)
Angiopteris evecta (G. Forst.) Marattiaceae Wan keep rat Rhizome Flavorless SKP110-10105 01 5.56 Reduce fever, use as astringent
Hoffm.
Cordyline fruticosa (L) Asparagaceae Mak mia Leaf Flavorless SKP005030601 5.56 Reduce fever, treat
Goeppert. (Green leaves) exanthematous fever and itch
Cordyline fruticosa (L) Asparagaceae Mak phu Leaf Flavorless SKP005030601 5.56 Reduce fever, treat
Goeppert (red leaves) exanthematous fever and itch
Cyathea gigantean Holtt. Cyatheaceae Ma has sa dam Stem Cool SKP059030701 5.56 Reduce fever and pain,
treat cough
Dracaena loureiri Gagnep. Dracaenaceae Chan deang Stem Bitter SKP065041201 5.56 Reduce fever, scurvy and
abscess
Eupatorium stoechadosmum Hance Compositae San phra hom Leaf Cool& SKP051051901 5.56 Treat fever, use as astringent
Flavorless
Globba malaccensis Ridl. Zingiberaceae Wan ron thong Rhizome Hot& Fragrant SKP206071301 5.56 Use as antiallergic, insect bites
Kaempferia galanga Linn Zingiberaceae Proh hom Rhizome Hot& Fragrant SKP206110701 5.56 Treat cold, use as carminative
Kheaw-Hom - - - Bitter& Cool - 100 Treat fever, measles,
chickenpox and aphthous ulcers
Limnophila rugosa Merr Scrophulariaceae Phak krachom Leaf Cool& Fragrant SKP177121801 5.56 Treat exanthematous fever
Mammea siamensis Kosterm. Guttiferae Sa ra phi Flower Cool& Fragrant SKP083131901 5.56 Cardiac tonic, treat vertigo
Mesua ferrea Linn. Guttiferae Bun nak Flower Cool& Fragrant SKP083130601 5.56 Cardiac tonic, treat vertigo
Mimusops elengi Linn. Sapotaceae Phi kul Flower Cool& Fragrant SKP171130501 5.56 Cardiac tonic, blood tonic
Myristica fragrans Houtt Myristicaceae Chan thet Stem Hot& Fragrant SKP121130601 5.56 Reduce fever, use as carmina
carminative
Nelumbo nucifera Gaertn. Nelumbonaceae Bua luang Pollen Astringent& SKP125141401 5.56 Cardiac tonic, treat vertigo
Fragrant
Pogostemon cablin Labiatae Phim sen thon Leaf Cool& Fragrant SKP095160301 5.56 Reduce fever, use as
(Blanco) Benth. carminative
Sophora exigua Craib Fabaceae Phit sa nat Trunk Bitter SKP072190501 5.56 Reduce fever, increase breast
milk
Tacca chantrieri Andre Taccaceae Nae ra phu sri Rhizome Astringent SKP189200301 5.56 Reduce fever, use as astringent
Vetiveria zizanioides (L.) Gramineae Faek hom Root Cool& Fragrant SKP081222601 5.56 Use as diuretic and carminative
Nash ex Small
Table 1. Plants and part of plant components in Kheaw-Hom remedy
S244 J Med Assoc Thai Vol. 100 Suppl. 5 2017
of 0.68-0.72 at 734 nm before use. Each extract (20 μL) at
the same concentration range mentioned above was
mixed with ABTS•+ solution (180 μL) and incubated at
room temperature for 6 min. The absorbance of these
concentrations was measured at 734 nm. The percent
of ABTS•+ scavenging activity in this concentration
range was calculated, and EC50 (μg/mL) was determined
using the method described above. Trolox was used as
a positive control. Experiments were done in triplicate.
The calculation of percent scavenging activity is by
the following formula:
% Inhihition = ((Mean of ODControl – Mean of ODsample)/
Mean of ODControl) x 100
Effective concentration of sample required to
inhibited ABTS radical by 50% (EC50) was obtained
by linear regression analysis of the dose-response
curve of % inhibition versus concentration, and EC50
is calculated using prism program. All determinations
were carried out in triplicate.
Intracellular superoxide anion scavenging
assay (NBT assay)(17)
Phorbol 12-myristate 13-acetate (PMA) was
used to stimulate differentiated HL-60 cells to generate
superoxide anions (O2•-) via respiratory burst, which
then reduced the nitroblue tetrazolium (NBT) solution
to blue formazan. Prior to performing the NBT assay,
the optimal concentration of sample with no cytotoxic
effects on HL-60 cells was determined using the MTT
assay. In NBT assay, differentiated HL-60 cells (1x106
cells) in Hank’s buffered salt solution (HBSS) (200 μl)
was incubated with each sample (500 μl) at the optimal
concentration for 15 min 37°C in a 5% CO2 atmosphere.
Next, the mixture was incubated for 60 min with a final
concentration of 250 ng/ml PMA and 0.625 mg/ml
NBT in HBSS. The reaction was stopped by adding
2 ml of 1 M HCl and centrifuged at 4,000 rpm for 10 min
to collect cell pellet containing formazan, which
was then dissolved in DMSO (300 μl). The absorbance
was determined at 572 nm. The O2•- scavenging activity
of the extract at the optimal concentration was
calculated:
Statistical analysis
All experiments were carried out in triplicate.
Statistical analysis was performed using Prism
Software.
(Mean of ODcontrol – Mean of ODbaseline)-(Mean of ODextract –Mean of ODbaseline)
Mean of ODcontrol – Mean of ODbaseline
% Inhibition =
Results
Antioxidant activity
The effect of the ethanolic and aqueous
extracts of Kheaw-Hom remedy and each of its herbal
components were studied using DPPH radical
scavenging assay, ABTS radical scavenging assay and
NBT assay. IC50 values are summarized in Table 2, 3.
Discussion
Kheaw-Hom remedy was used in Thai
traditional medicine as antipyretic for exanthematous
fever such as measles and chickenpox. Kheaw-Hom
remedy was reported in the previous studies on several
biological activities such as antiviral, anti-inflammatory
and antimicrobial. Firstly, antiviral activity against
enterovirus 71 (EV71) which cause hand, foot and
mouth disease that its aqueous extract at concentration
of 400 μg/ml inhibited EV71 concentrate 25TCID50(18).
Secondly, anti-inflammatory activity by inhibiting nitric
oxide release in RAW 264.7 that the aqueous and
ethanolic extracts had weak activity showed IC50 value
of 48.86 and 59.77 μg/mL, respectively(18). Lastly, the
ethanolic extract had antimicrobial activity against three
gram-positive bacteria of skin infection complications
in exanthematous fever include Staphylococcus aureus,
Methicillin-resistant Staphylococcus aureus (MRSA),
Staphylococcus epidermis with an inhibition zone
of 7.33, 7.00, 8.00 mm, respectively; yet showed no
inhibition on fungus (Candida albicans)(19). This study
is the first report on antioxidant activity of Kheaw-
Hom remedy using DPPH and ABTS radical scavenging
assays that the ethanolic extract had strong activity in
DPPH radical scavenging assay and had weak activity
in ABTS radical scavenging assay. The aqueous extract
had weak activity in ABTS radical scavenging assay
and had no activity on DPPH radical scavenging assay.
All these results could support use of Kheaw-Hom
remedy related with Thai traditional medicine used for
exanthemathous fever.
There are nine plant ingredients in
Kheaw-Hom remedy that were reported in the previous
study on antioxidant activity using DPPH radical
scavenging assay including P. cablin, C. fruticosa,
V. zizanioides, M. fragrans, G. malaccensis, C.
gigantean, M. elengi, M. siamensis and N. nucifera.
Firstly, the previous study demonstrated the antioxidant
activity using DPPH radical scavenging assay and the
methanolic extract of N. nucifera(12) and vetiver oil of
V. zizanioides(14) dissolved in methanol had strong
activity and, this present study showed that the
aqueous extract of N. nucifera had strong activity but,
J Med Assoc Thai Vol. 100 Suppl. 5 2017 S245
Botanical name Code Antioxidant activity
DPPH scavenging assay ABTS scavenging assay NBT dye reduction assay
(EC50 + SEM) μg/mL (EC50 + SEM) μg/mL (IC50 + SEM) μg/mL
Angiopteris erect (G. Forst) Hoffm. AEE 42.95+4.24 >100 >100
Cordyline fruticosa (L.) Goeppert. (green leaves) CGE >100 >100 >100
Cordyline fruticosa (L.) Goeppert. (red leaves) CRE 47.55+4.448 >100 >100
Cyathea gigantea Holtt. CyGE 7.55+0.893 20.09+2.960 >100
Dracaena loureiri Gagnep. DLE 13.89+1.138 7.88+0.650 >100
Eupatorium stoechadosmum Hance ESE 50.97+1.187 >100 >100
Globba malaccensis Ridl. GM E 61.29+4.982 8.06+0.53 >100
Kaempferia galanga Linn. KGE >100 >100 >100
Kheaw-Hom remedy KHE 16.96+1.214 30.91+1.530 >100
Limnophila rugosa Merr. LRE >100 >100 >100
Mammea siamensis Kosterm MSE 36.60+5.030 71.86+3.250 >100
Mesua ferrea L. MeFE 37.40+1.954 63.15+4.033 >100
Mimusops elengi L. MEE 53.89+0.645 >100 >100
Myristica fragrans Houtt MFE >100 >100 >100
Nelumbo nucifera Gaertn. NUE >100 >100 >100
Pogostemon cablin (Blanco)Benth. PCE 90.90+5.029 >100 >100
Sophora exigua Craib SEE 9.42+2.107 >100 >100
Tacca chantrieri Andre TCE 8.00+2.368 14.84+0.48 63.38+3.290
Vetiveria zizanioides (L.) Nash ex Small VZE >100 >100 >100
BHT 13.40+0.266 - -
Trolox - 5.850+0.730 -
Propyl gallate - 14.87+0.02
Table 2. Antioxidant activities on DPPH scavenging assay, ABTS scavenging assay and NBT dye reduction assay of ethanolic extract of Kheaw-Hom remedy and its plant
ingredients
S246 J Med Assoc Thai Vol. 100 Suppl. 5 2017
Botanical name Code Antioxidant Activity
DPPH scavenging assay ABTS scavenging assay NBT dye reduction assay
(EC50+SEM) μg/mL (EC50+SEM) μ g/mL (IC50+SEM) μg/mL
Angiopteris evecta (G.Forst) Hoffm. AEW >100 >100 >100
Cordyline fruticosa (L.) Goeppert. (green leaves) CGW >100 >100 >100
Cordyline fruticosa (L.) Goeppert. (red leaves) CRW >100 >100 >100
Cyathea gigantea Holtt. CyGW 14.40+2.07 20.16+1.51 >100
Dracaena loureiri Gagnep. DLW 10.00+0.88 15.31+4.09 >100
Eupatorium stoechadosmum Hance ESW >100 82.06+3.17 >100
Globba malaccensis Ridl. GMW >100 >100 >100
Kaempferia galanga Linn. KGW >100 >100 >100
Kheaw-Hom remedy KHW >100 64.89+0.82 >100
Limnophila rugosa Merr. LRW >100 >100 >100
Mammea siamensis Kosterm MSW 11.45+1.24 23.10+1.20 >100
Mesua ferrea L. MeFW 15.91+4.69 18.30+1.27 >100
Mimusops elengi L. MEW 32.56+2.25 24.24+2.58 >100
Myristica fragrans Houtt MFW 25.81+0.37 21.21+1.17 >100
Nelumbo nucifera Gaertn. NUW 15.68+2.46 19.33+0.97 >100
Pogostemon cablin (Blanco) Benth. PCW 18.13+2.35 37.31+1.31 >100
Sophora exigua Craib SEW >100 74.07+4.30 >100
Tacca chantrieri Andre TCW 9.46+1.48 16.00+0.44 70.65+1.28
Vetiveria zizanioides (L.) Nash ex Small VZW >100 >100 >100
BHT 13.40+0.266 - -
Trolox 5.85+0.73 -
Propyl gallate - 14.87+0.02
Table 3. Antioxidant activities on DPPH scavenging assay, ABTS scavenging assay and NBT dye reduction assay of aqueous extract of Kheaw-Hom remedy and its plant
ingredients
J Med Assoc Thai Vol. 100 Suppl. 5 2017 S247
that of V. zizanioides had no activity. Secondly, the
ethanolic extract of P. cablin(20), the ethanolic and
aqueous extracts of M. fragrans(21), the ethanolic extract
of bark of C. gigantean(24) and methanolic extract of
flower of M. elengi(9) had moderate activity and, in this
present study the ethanolic extract of P. cablin had
weak activity, only the aqueous extract of M. fragrans
had moderate activity, the ethanolic extract of C.
gigantean had strong activity and the aqueous extract
of M. elengi had moderate activity. Finally, the aqueous
extract of P. cablin(20), the methanolic extract of C.
fruticosa(23) and the ethanolic extract of G.
malaccensis(24) had weak activity, and aqueous extract
of P. cablin had strong activity, the ethanolic extract of
of C. fruticosa had moderate activity and the ethanolic
extract of G. malaccensis had weak activity. The results
showed different values may be from the usage various
solvent in extraction and source of plants lead to the
chemical constituents of plants were different.
Interestingly, the ethanolic and aqueous
extracts of T. chantrieri showed strong efficacy in
DPPH and ABTS radical scavenging assays and had
weak efficacy in NBT assay. T. chantrieri was reported
in the previous study, ABTS radical scavenging assay
that ethanolic extract had moderate efficacy(25), but for
DPPH radical scavenging assay and NBT assay have
never been reported.
Conclusion
To the best of our knowledge, the best
antioxidant activity using DPPH scavenging assay are
that of ethanolic extract of C. gigantean, T. chantrieri
and S. exigua having EC50 +SEM values of 7.55+0.89,
8.00+2.37 and 9.42+2.11 μg/mL, respectively. The best
antioxidant activity using ABTS scavenging assay are
that of ethanolic extract of D. loureiri, G. malaccensis
and T. chantrieri having EC50 +SEM values of
7.88+0.65, 8.06+0.53 and 14.84+0.48 μg/mL,
respectively.
The best antioxidant activity using NBT dye
reduction assay are that of ethanolic and aqueous
extracts of T. chantrieri have IC50 +SEM values of
63.38+3.29 and 70.65+1.28 μg/mL, respectively.
Acknowledgements
This work was supported by the National
Research University Project of Thailand Office of
Higher Education Commission and Center of
Excellence on Applied Thai Traditional Medicine
Research (CEATMR). Faculty of medicine, Thammasat
University.
Potential conflicts of interest
None.
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J Med Assoc Thai Vol. 100 Suppl. 5 2017 S249
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