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DPPH Scavenging Activities and Phytochemical Content of Four Asteraceae

Plants

Article in International Journal of Pharmacognosy and Phytochemical Research · June 2017

DOI: 10.25258/phyto.v9i6.8173

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International Journal of Pharmacognosy and Phytochemical Research 2017; 9(6); 755-759
DOI number: 10.25258/phyto.v9i6.8173
ISSN: 0975-4873
Research Article

DPPH Scavenging Activities and Phytochemical Content of Four

Asteraceae Plants
Raden Bayu Indradi, Irda Fidrianny, Komar Ruslan Wirasutisna
Pharmaceutical Biology Research Group, School of Pharmacy, Bandung Institute of Technology, Indonesia

Received: 1st April, 17; Revised 20th May, 17, Accepted: 12th June, 17; Available Online:25th June, 2017

ABSTRACT
Free radicals are atoms or molecules that have one or more unpaired electrons on its outer orbital, highly reactive, and
could damage cell inside human body. Human body produce antioxidant to neutralize free radicals, but human ageing
and stress oxidative conditions would increase the formation of free radicals, therefore an exogenous antioxidant are
needed. Asteraceae family is the largest family among the plant kingdom therefore it has great potential as source of
exogeneous antioxidants. The objectives of this research were to determine antioxidant activities of aerial part of
elephant’s foot (Elephantopus scaber L.), false daisy (Eclipta alba (L.) Hassk.), Indian pluchea (Pluchea indica (L.)
Less), and dandelion (Taraxacum officinale Weber ex F.H Wigg.) using DPPH method, determine total flavonoid and
total phenolic content, and analyze correlation between total flavonoid content and total phenolic content with
antioxidant activity. Extraction was carried out by reflux with increasing polarity using n-hexane, ethyl acetate, and
ethanol respectively. Antioxidant activity was determined by DPPH method. Total flavonoid content was determined
using Chang’s method and total phenolic content evaluated using Folin—Ciocalteu reagent. Correlation of total
flavonoid content and total phenolic content was analyzed by Pearson’s method. Ethanolic extract of Indian pluchea
showed the highest antioxidant activity with IC50 DPPH 16.66 ± 0.08 µg/mL. The highest total phenolic content (23.49 ±
0,56 g QE (Quercetin Equivalent)/100 g) was given by ethyl acetate extract of Indian pluchea, while the highest
flavonoid content (16.48 ± 0.25 g GAE (Gallic Acid Equivalent)/100 g) was showed by ethanolic extract of Indian
pluchea. Total phenolic content of elephant’s foot, false daisy and Indian pluchea herbs extracts showed significantly
negative correlation with their IC50 of DPPH scavenging activities. Indian pluchea herbs extract had the highest
antioxidant activity using DPPH method compared to elephant’s foot, false daisy and dandelion herbs. Phenolic
compounds were the major contributor in antioxidant activities of elephant’s foot, false daisy and dandelion herbs
extracts by DPPH method.

Keywords: Antioxidant, DPPH, Asteraceae, Pluchea indica.

INTRODUCTION activity6, hydrogen peroxide scavenging assay6, and in

Free radicals are atoms or molecules that have one or rat liver7. Ethanolic extract of Eclipta alba showed
more unpaired electrons on its outer orbital, highly antioxidant activity in DPPH scavenging8 and other
reactive, and could damage cell inside human body. several methods9. Ethanolic extract of Elephantopus
Human body produce antioxidant to neutralize free scaber showed antioxidant activity in superoxide
radicals, but human ageing and stress oxidative scavenging activity, lipid peroxidase inhibition, and
conditions would increase the formation of free hydroxyl radical scavenging methods10. Methanolic
radicals, therefore an exogenous antioxidant are extract of Taraxacum officinale showed antioxidant
needed1. Asteraceae is the largest family among the activity with DPPH scavenging11 and FRAP
plant kingdom, therefore it has a great potential as methods11,12. There was no research regarding
source of exogenous antioxidant. Elephant’s foot comparison of antioxidant activity using Elephantopus
(Elephantopus scaber L.), false daisy (Eclipta alba (L.) scaber, Eclipta alba, Pluchea indica, and Taraxacum
Hassk.), Indian pluchea (Pluchea indica (L.) Less.), and officinale herbs which was extracted by increasing
dandelion (Taraxacum officinale Weber ex F.H Wigg) polarity solvent.
are some Asteraceae plants that thrives widely in
Indonesia. Previous research showed that ethanolic and MATERIAL AND METHODS
methanolic extract of Pluchea indica gave an Materials
antioxidant activity with DPPH scavenging method2,3. DPPH (2,2- diphenyl - 1 picrylhydrazyl), gallic acid,
Methanolic extract of Eclipta alba showed antioxidant quercetin, ascorbic acid were purchased from Sigma-
activity with DPPH scavenging method4,5,6, nitric oxide Aldrich (MO, USA), Elephantopus scaber, Eclipta
radical scavenging assay6, lipid peroxidation inhibitory alba, Pluchea indica, Taraxacum officinale, methanol,

*Author for Correspondence: [email protected]

 Raden et al. / DPPH Scavenging Activities…

ethanol, ethyl acetate, n-hexane, and all other analytical All dried samples were extracted by reflux. Each
materials used in the study. sample was extracted with n-hexane then the residue
Sample Preparations was dried and further extracted with ethyl acetate and
Elephantopus scaber, Eclipta alba, Pluchea indica, and the residue was dried and lastly extracted by ethanol.
Taraxacum officinale herbs were freshly collected from All extraction process was performed in triplicate.
Lembang, West Java – Indonesia and determined in Extracts were concentrated by rotary evaporator. Yield
Herbarium Bandungense, School of Life Sciences and of each extract was determined and could be seen in
Technology, Bandung Institute of Technology. All Table 1.
samples were sorted, washed, dried at 40 0-500 C and Total flavonoid content
grinded into powder. Total flavonoid content of all extract could be seen in
Extraction Fig 1. The results were varied from 0.83 to 23.49 g
All samples were extracted by reflux, using increasing QE/100 g. The total flavonoid content of all 12 extract
polarity solvents which were n-hexane, ethyl acetate, were obtained by using standard calibration curve of
ethanol respectively. All extraction was performed in quercetin y = 0.0094x - 0.1573, R2 = 0.9987.
triplicate. All extracts were concentrated by rotary Total phenolic content
evaporator resulted n-hexane, ethyl acetate, and ethanol Total phenolic content of all extract could be seen in
extracts of Elephantopus scaber (ES1), (ES2), (ES3), Fig 2. The results were varied from 3.19 to 16.48 g
Eclipta alba (EA1), (EA2), (EA3), Pluchea indica GAE/100 g. The total phenolic content of all 12 extracts
(PI1), (PI2), (PI3) and Taraxacum officinale (TO1), were obtained by using standard calibration curve of
(TO2), (TO3). gallic acid y = 0.0051x – 0.1662, R2 = 0.9971.
Total flavonoid content Antoxidant Activity
Total flavonoid content was conducted by Chang et al. IC50 of DPPH scavenging activities of 12 extracts could
method13. Quercetin was used as standard. Aquadest 2.8 be seen in Fig 3. The results were widely varied,
ml was added with 1.5 ml methanol, 0.1 ml aluminium ranging from 16.66 to 1109.17 µg/ml, while IC50 of
chloride 10%, 0.1 ml sodium acetate 1 M and 0.5 ml DPPH ascorbic acid as standard was 1.83 µg/ml
extract in methanol. The mixture was further incubated Correlation of total flavonoid and phenolic content with
for 30 minutes in room temperature. Absorbance was antioxidant activity
measured by UV-visible spectrophotometry at λ 415 Correlation of total flavonoid and phenolic content with
nm. their IC50 of DPPH scavenging activities showed that
Total phenolic content total phenolic content of Elephantopus scaber, Eclipta
Total phenolic content determination was done using alba, and Pluchea indica gave a negative and
Folin-Ciocalteu reagent. Gallic acid was used as significant correlation with their IC50 of DPPH
standard. Each extract was dissolved in methanol then scavenging activities, while total flavonoid content
0.5 ml extract was added by 5 ml Folin-Ciocalteu showed no significant correlation with their IC50 of
reagent and 4 ml sodium carbonate 1 M, then incubated DPPH scavenging activities.
for 15 minutes in room temperature. Absorbance was
measured by UV-visible spectrophotometry at λ 765 DISCUSSION
nm14. Extraction with increasing polarity solvent was done to
Antioxidant Activity achieve optimum yield of all polarity (non polar, semi
Antioxidant activity was done by Molyneux method15. polar, and polar) extracts. Firstly extraction was carried
Methanol was used as blank, DPPH 50 µg/mL as out with n-hexane to extract non polar compounds. The
control and ascorbic acid as standard. Two ml of second extraction was conducted using ethyl acetate to
standard or sample was prepared in various the residue which extracted semi polar compounds. The
concentration then added into 2 ml DPPH 50 µg/ml last extraction was performed with ethanol to the
solution and incubated for 30 min before measured by residue which extracted polar compunds. The yield of
UV-visible spectrophotometry at λ 515 nm and the extracts could be seen in Table 1. Ethanolic extract
performed triplicate. IC50 of sample or standard was of Taraxacum officinale (TO3) showed the highest
calculated by DPPH scavenging activity calibration yield with 13.26%. Ethanolic extract showed the
curve16. highest yield of all plants compared to n-hexane and
Statistical Analysis ethyl acetate, could be indicate that majority compound
Each analysis was performed triplicate and data of all plants were polar compounds.
processing was done by SPSS software. The result Total flavonoid content in ethyl acetate herbs extract of
presented as mean ± deviation standard using ANOVA Pluchea inidica (PI2) was the highest with 23.49 g
and Tukey post-hoc (p<0.05). Correlation analysis QE/100 g followed by n-hexane herbs extract of
between flavonoid content and phenolic content with Pluchea indica (PI1), ethyl acetate herbs extract of
antioxidant activity was conducted by Pearson’s Eclipta alba (EA2), and ethyl acetate herbs extract of
method17. Elephantopus scaber (ES2) (16.58, 16.02, 12.02 g
QE/100 g, respectively). AlCl3 will form a complex
RESULT with the OH of flavonoid compounds at C3’-C4’, and or
Extraction OH at C-3 and 4 oxo, and or OH at C-5 and 4 oxo18. It

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 Raden et al. / DPPH Scavenging Activities…

Table 1: Yield of various extracts.

Yield (% w/w)
Extract
Elephantopus scaber Eclipta alba Pluchea indica Taraxacum officinale
n-Hexane 3.20 1.45 3.26 2.55
Ethyl acetate 3.11 2.80 2.23 2.27
Ethanol 7.70 8.92 11.31 13.26

Figure 1: Total flavonoid content of all samples. Figure 2: Total phenolic content of all samples.

Figure 3: IC50 of DPPH scavenging activity of all samples and standard.

g) compared to the other extracts, followed by PI2,

ethanolic herbs extract of Elephantopus scaber (ES3)
Table 2: Correlation of total flavonoid and phenolic content with IC 50 DPPH scavenging activity.
Pearson’s Correlation Coefficient (r)
Antioxidant Parameter
Total Flavonoid Total Phenolic
IC50 DPPH Elephantopus scaber -0.079ns -0.952**
IC50 DPPH Eclipta alba -0.402ns -0.992**
IC50 DPPH Pluchea indica 0.353ns -0.819**
IC50 DPPH Taraxacum officinale 0.166ns -0.180ns
ns: not significant, **: significant at p<0.01

can be inferred that many flavonoid compounds was and PI1 (9.48, 8.74, 6.48 g GAE/100 g, respectively).
semipolar flavonoid which soluble in ethyl acetate Unlike the flavonoids, phenolic compound was highest
extract and less in ethanolic extract, which indicated the in ethanolic extracts compared to n-hexane and ethyl
polarity range of flavonoid in these plants. This acetate except for Taraxacum officinale which the ethyl
flavonoid might have some hydroxyl (-OH) and methyl acetate extract contain higher phenolic compound than
(-CH3) substitution. ethanolic extract. Phenolic compound are well known
Total phenolic compound in ethanolic herbs extract of as polar compound, therefore it’s likely to be extracted
Pluche indica (PI3) was the highest (16.48 g GAE/100 in polar solvent but not all phenolic are polar

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 Raden et al. / DPPH Scavenging Activities…

compound. Any compound with hydroxyl substitution therefore phenolic compounds were clearly the major
at benzene structure is considered as phenolic contributors of antioxidant activity. It was similar to
compound, and in the same structure there might be EA3 and EA2, thus confirmed phenolic compounds as
another substitution such as methyl (-CH3) that can major contributors of their antioxidant activity.
decrease polarity of a compound. This reason could Overall, Pluchea indica showed the greatest antioxidant
explain how total phenolic compound of TO2 is higher activity among others. Srimoon and Ngiewthaisong 2
than TO3. previously reported that dried stem part of Pluchea
Antioxidant activity was done by DPPH scavenging indica showed the highest antioxidant activity and also
method. DPPH is a stable radical, which in its radical had the highest total phenolic content among other parts
form gives a violet color15. Antioxidant will react with of plants, while Noridayu et al. 21 exposed that
DPPH by electron donate mechanism, which stabilized methanolic leaves extract of Pluchea indica showed
DPPH was demonstrated by decreasing intensity of higher antioxidant and total phenolic compound than
DPPH’s violet color and slowly turns into yellow and metanolic stem extract. Widyawati et al. 3 presented
this decrease could be measured by visible that antioxidant activities of Pluchea indica leaves
spectrophotometry at λ 515nm19. The highest which was extracted by gradient polarity solvent from
antioxidant activity was showed by PI3 with IC50 DPPH n-hexane to water exhibited that methanolic extract
scavenging activity of 16.66 μg/ml followed by showed the highest total phenolic content, total
ethanolic herbs extract of Elephantopus scaber (ES3) flavonoid content, and also the highest antioxidant
18.43 μg/ml, ethanolic herbs extract of Eclipta alba activity with DPPH scavenging method slightly better
(EA3) 23.79 μg/ml, and ethyl acetate herbs extract of than water and ethanolic extracts. It was similar to the
Eclipta alba (EA2) 24.33 μg/ml. The data in Fig 3 present study, ethanolic extract of Pluchea indica had
indicated that ethanolic extract of each plant gave the the greatest antioxidant activity of all extracts,
highest antioxidant activities compared to n-hexane and the ethyl acetate extract was the second after Eclipta
ethyl acetate extracts. alba among other ethyl acetate extracts, and n-hexane
Pearson’s correlation was performed to see the extract was the greatest among other n-hexane extracts
correlation of total flavonoid and phenolic content with from other plants.
their antioxidant activities. The result in Table 2
showed that total phenolic content in Elephantopus CONCLUSION
scaber, Eclipta alba, and Pluchea indica herbs extracts Ethanolic extract of Pluchea indica herbs (PI3) showed
had significantly negative correlation with their IC50 the highest antioxidant activity with IC50 DPPH
DPPH scavenging activities. Negative correlation scavenging activity of 16.66 µg/ml. The highest total
means the higher value of total flavonoid/total phenolic flavonoid content was given by ethyl acetate herbs
content will give lower IC50 DPPH scavenging activity extract of Pluchea indica (PI2) 23.49 g QE/ 100 g and
value. The lower IC50 value means stronger antioxidant highest total phenolic content was given by ethanolic
activity. No significant correlation of total flavonoid herbs extract of Pluchea indica (PI3) 16.48 g GAE/100
with their IC50 DPPH value can be explained as not all g. Total phenolic content in Elephantopus scaber,
flavonoid give a strong antioxidant property. Flavonoid Eclipta alba, and Pluchea indica herbs extracts gave
with di -OH substitution at C3’ and C4’ gives the significantly negative correlation with their IC50 DPPH
strongest antioxidant properties, followed by C2-C3 scavenging activities.
double bond, and –OH substitution at C3. As showed in
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