The Indonesian Biomedical Journal.
Vol.
No.
August 2017, p.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179
RESEARCH ARTICLE
Antioxidant Activity of Phyllanthus niruri Extract.
Rutin and Quercetin Djaja Rusmana1.
Roro Wahyudianingsih1.
Mariska Elisabeth1.
Balqis2.
Maesaroh2.
Wahyu Widowati1,E Faculty of Medicine.
Maranatha Christian University.
Jl.
Prof.
Suria Sumantri No.
65 Bandung 40164.
Indonesia Biomolecular and Biomedical Research Center.
Aretha Medika Utama.
Jl.
Babakan Jeruk 2 No.
Bandung, 40163.
Indonesia Corresponding author.
E-mail: wahyu_w60@yahoo.
Received date: Nov 29, 2016.
Revised date: Feb 20, 2017.
Accepted date: May 3, 2017 Abstract ACKGROUND: Normal metabolism of oxygen and exogenous factors constantly generate free radicals which could be harmful to the human Human need antioxidants to provide protection against free radicals, thus plants are a good source of natural antioxidants.
Phyllanthus niruri (P.
has been known to possess several medicinal properties and contain numerous active phytochemical.
In this research, we conducted phytochemical screening and antioxidant assay of P.
niruri extract along with the compounds rutin and quercetin, which are flavonoids possessing medicinal This study was conducted to determine P.
rutin and quercetin as antioxidant.
METHODS: In this study, qualitative phytochemical screening was performed to detect phenol, flavonoid, saponin, tannin, steroid/triterpenoid, terpenoid and alkaloid in P.
niruri extract.
Antioxidant analysis of P.
niruri, rutin and quercetin was conducted using total Introduction Reactive Oxygen Species (ROS) leads to damaging the lipids in the cell membranes, proteins in tissues as well as enzymes, carbohydrates, and DNA to induce oxidation.
This oxidative damage may play a causative role in aging and several diseases which are cancer, cardiovascular disease, cataracts, and cognitive dysfunction.
The high ROS level plays a role in oxidative stress.
Therefore, the measured phenolic content, 2,2-diphenyl-1-picrylhydrazil (DPPH), 2,2Ao-azinobis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) RESULTS: The study revealed that P.
niruri extract contained saponin, phenol, flavonoid and tannin based on phytochemical screening.
In DPPH and ABTS assays quercetin possessed highest antioxidant activity with IC50 value of 0.
55 and 1.
17 AAg/ml respectively.
Meanwhile.
niruri extract showed the highest FRAP activity which was 95 AAM Fe(II)/AAg extract.
Rutin possessed the lowest antioxidant activity in all antioxidant assays.
CONCLUSION: This study confirmed that P.
niruri extract and quercetin have great potential as a natural antioxidant KEYWORDS: asntioxidant, phytochemical.
Phyllanthus niruri, quercetin, rutin, free radical Indones Biomed J.
: 84-90
body requires exogenous antioxidant supply to prevent the oxidative stress.
The dietary antioxidant can be obtained naturally from plants or manufactured synthetically, but most of the natural antioxidants have better antioxidant activity compared with the synthetic one and are considerably safer.
Various compounds which have antioxidant activity have been isolated from plants, many of them are polyphenols including phenols, phenolic acids, flavonoids, tannins, and .
DOI: 10.
18585/inabj.
Antioxidant of Phyllantus niruri Extract.
Rutin, and Quercetin(Rusmana D, et a.
Indones Biomed J.
: 84-90
Phyllanthus niruri (P.
is one of the herbal medicinal plants that have a broad range of properties, which are antiviral activities against hepatitis B, antimicrobial, hepatoprotective, anticancer, and hypocalcemic agent.
Several active phytochemicals have been discovered in P.
niruri, such as flavonoids, alkaloids, terpenoids, lignans, polyphenols, tannins, coumarins, and saponins.
A number of flavonoids had been successfully identified from P.
including rutin and quercetin, which are well known to have significant antioxidants and chelating properties.
In the present study, antioxidant potential of P.
niruri was investigated through 2,2Ao-azinobis-3-ethylbenzo-thiazoline6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazil (DPPH) and ferric reducing antioxidant power (FRAP) activity assays and also qualitative phytochemical screening assay with two flavonoid compounds rutin and quercetin for comparison.
Protective antioxidant by plant products properties such as P.
niruri may contribute as therapeutic drugs for free radical induced pathologies.
flavonoid was shown by the formation of red or orange Alkaloid identification: the presence of yellow color indicated the positive result or presence alkaloid.
Total Phenol Content Assay Total phenol content was determined using Folin-Ciocalteu .
First, 15 AAL samples were introduced into a 96-well microplate.
Subsequently, 75 AAL of Folin-Ciocalteu reagent .
%) and 60 AAL of sodium carbonate .
5%) were The plate was shaken and incubated at 50oC for 10 mins.
Finally, absorbance value was measured using a microplate reader at the wavelengths of 760 nm.
The total phenol content was expressed as Gallic Acid equivalent (GAE).
Quercetin equivalent (QE), and Rutin equivalent (RE) using the following formula:
C = .
x V) / m C: total content of phenolic compounds (AAg/mg P.
niruri extract in GAE.
QE, or RE).
c: concentration of GAE.
QE, or RE calculated from the regression Methods Preparation of P.
niruri Extract The stem and leaves of P.
niruri were collected from Cianjur.
West Java.
Indonesia in February 2015.
The plants were identified by herbarium staff.
Department of Biology.
School of Life Science and Technology.
Bandung Institute of Technology.
Bandung.
Indonesia.
The stem and leaves of P.
niruri were mashed, extracted using ethanol 70% by a maceration method.
The ethanol filtrate was filtered every 24 hours and collected until it was colorless.
Evaporation was conducted to get concentrated ethanol extract in paste The extract was stored at -20oC.
Qualitative Phytochemical Screening Assay The phytochemical assay was conducted on P.
extract using modified Fransworth method to qualitatively identified the presence of phytochemicals.
,15-.
Phenol identification: the presence of phenol indicated by green/red/purple/blue or black color.
Steroid/triterpenoid identification: the formation of green/blue color indicates the presence of steroid while red/orange sediment indicates the presence of triterpenoid.
Saponin identification: saponin content was indicated by persistence of froth on the surface.
Tannin identification: purple color formation indicates positive reaction for tannin.
Terpenoid identification:
terpenoid was indicated by the formation of purple color in the mixture.
Flavonoid identification: the presence of equation (AAg/mL).
V: volume of extract .
L).
m: weight of P.
niruri extract .
DPPH Assay Briefly, 50 AAL of samples were added to each well in a 96well microplate.
Then it was followed by adding 200 AAL of DPPH (D9132.
Sigma-Aldrich.
St.
Louis.
MO.
USA) solution .
077 mmol/L in methano.
into the well.
The mixture then was incubated in the dark for 30 mins at room Afterward, the absorbance was read using a microplate reader at 517 nm.
,12,19,.
The radical scavenging activity was measured with following formula:
Scavenging % = (Ac Ae A.
/ Ac x 100 Ac: negative control absorbance .
ithout sampl.
As: sample absorbance ABTS-reducing Activity Assay ABTS solution was produced by reacting 14 mM ABTS (A1888.
Sigma-Aldrich.
St.
Louis.
MO.
USA) and 4.
9 mM calcium persulfate .
:1 volume rati.
for 16 hours in dark condition at room temperature, then the mixture was diluted with phosphate-buffered saline (PBS) .
H 7.
until the absorbance of the solution was 0.
70 A 0.
02 at wavelengths 745 nm.
In brief, 2 AAL of samples were added to each well at 96-well microplate, then fresh 198 AAL ABTS solution were The absorbance was measured at 745 nm after the plate had been incubated for 6 min at 30oC.
The percentage The Indonesian Biomedical Journal.
Vol.
No.
August 2017, p.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179 inhibition of ABTS radical (%) was determined by the ratio of reducing of ABTS absorbance in the presence of the sample relative to the absorbance in the absence of the sample .
egative contro.
The median inhibitory concentration (IC.
were also calculated.
Table 1.
Qualitative phytochemical screening results of P.
FRAP Assay The FRAP reagent was prepared freshly by mixing 10 mL of 300 mM acetate buffer .
H 3.
6 adjusted with the addition of acetic aci.
and 1 mL of 20 mM ferric chloride hexahydrate (Merck 1.
, and 1 mL of 10 mM 2,4,6-Tris.
-pyridy.
-s-triazine (TPTZ) (T1253.
SigmaAldrich.
St.
Louis.
MO.
USA).
In 96-well microplate, 7.
AAL of samples were mixed with 142.
5 AAL FRAP reagent then incubated for 30 min at 37oC.
The absorbance value was measured at 593 nm with a microplate reader (Thermo Scientific Multiscan GO.
Thermo Fisher Scientific.
Ratastie.
Finlan.
,18,.
Statistical Analysis Each experiment was conducted in a triplicate manner.
Data were expressed as a mean A standard deviation.
The differences between groups were statistically analyzed using one-way analysis of variance .
ne way ANOVA) with SPSS Statistics version 17.
0 software.
The probability values of p<0.
05 were considered as statistically significant using DuncanAos post hoc test.
Results Phytochemical Screening of P.
niruri Extract The qualitative phytochemical screening assay on P.
extract showed that the extract had a high content of saponins ( ), a moderate content of phenols, flavonoids, and tannins ( ).
It also had a low content of steroids, but triterpenoids, terpenoids, and alkaloids were not detected in the screening (-) (Table .
Total Phenol Content (TPC) The total phenolic content of P.
niruri extract was measured using GAE.
QE and RE as standard.
Total phenol content of P.
niruri extracts has value 61.
36 A 0.
42 AAg GAE/mg extract, 54.
72 A 0.
39 AAg QE/mg extrac.
29 A 2.
AAg RE/mg extract.
Those compounds were used since both are phenolic compounds that are known to be found in P.
The result revealed that total phenol content of P.
niruri extract was high, and the highest value was achieved by rutin as standard.
Phytochemical content niruri extract Phenols Steroids/ Triterpenoids /- Terpenoids Saponins Flavonoids Tannins Alkaloids .
ery high conten.
igh conten.
oderate conten.
ess conten.
- .
ot detecte.
DPPH Scavenging Activity The scavenging activity was increased along with the increase of the concentration of sample used.
It can be seen at the highest concentration among samples, quercetin exhibited the highest DPPH scavenging activity, followed by P.
niruri extract and the lowest was rutin (Table .
Quercetin had the lowest IC50 value followed by P.
niruri extract then rutin (Table .
The low IC50 suggested a high antioxidant activity, therefore quercetin has the most effective antioxidant activity in this DPPH assay.
ABTS-reducing Activity Among the samples, it can be seen that both P.
niruri extract and quercetin possess great ABTS-reducing activity, with the highest value in P.
niruri extract and quercetin were similar and statistically not significant.
Rutin had the lowest ABTS-reducing activities among three samples (Table .
The value of IC50 of P.
niruri extract, rutin, and quercetin in reducing the ABTS free radical in Table 5 revealed that niruri extract have low value of IC50, even though the value was still slightly higher than quercetin.
This assured that P.
niruri extract exhibited effective antioxidant activity but still behind quercetin.
Rutin was seen to have high IC50, indicating this compound was not effective in reducing the ABTS radical.
FRAP Activity The FRAP activity in this study showed that the activity was increased significantly in a concentration-dependent manner (Table .
Both P.
niruri and quercetin expressed high FRAP activity which indicates great antioxidant capability, while rutin remained to show the lowest activity.
At low concentration, quercetin showed higher FRAP activity than niruri, but at the highest concentration.
FRAP activity of niruri was comparable to quercetin.
Antioxidant of Phyllantus niruri Extract.
Rutin, and Quercetin(Rusmana D, et a.
Indones Biomed J.
: 84-90
DOI: 10.
18585/inabj.
Table 2.
DPPH scavenging activity of P.
niruri extract, rutin, and quercetin.
DPPH Scavenging Activity (%) Concentration (AAg/m.
niruri extract 94 A 0.
Rutin Quercetin 85 A 0.
96 A 0.
37 A 0.
05 A 0.
20 A 0.
74 A 0.
85 A 0.
59 A 0.
64 A 2.
60 A 0.
43 A 0.
96 A 0.
78 A 0.
54 A 0.
41 A 0.
83 A 0.
01 A 0.
72 A 2.
86 A 0.
77 A 0.
54 A 0.
40 A 0.
16 A 0.
*The data was presented as mean A standard deviation.
Different superscript small letters in the same column .
mong concentrations of the sample.
and capital letters in the same row .
mong samples in various concentratio.
indicate significance at p <0.
05 (Duncan post hoc tes.
Table 3.
IC50 DPPH scavenging activity of P.
niruri extract, rutin, and quercetin.
Samples niruri extract Rutin Quercetin Linear equation IC50 (AAg/m.
y = 7.
y = 7.
y = 7.
y = 6.
y = 6.
y = 6.
y = 57.
y = 57.
y = 57.
Average IC50 (AAg/m.
24 A 0.
56 A 0.
55 A 0.
*The data was presented as mean A standard deviation of IC50 .
The most active DPPH scavenging activity was quercetin with the lowest value of IC50 Table 4.
ABTS-reducing activity of P.
niruri extract, rutin, and quercetin.
Concentration (AAg/m.
ABTS-reducing Activity (%) niruri extract Rutin Quercetin 30 A 0.
72 A 1.
86 A 0.
31 A 0.
17 A 0.
33 A 0.
06 A 1.
78 A 0.
59 A 2.
56 A 1.
81 A 0.
60 A 1.
18 A 2.
94 A 0.
93 A 0.
76 A 0.
76 A 0.
55 A 0.
10 A 0.
08 A 0.
55 A 0.
*The data was presented as mean A standard deviation.
Different superscript small letters in the same column .
mong concentrations of the sample.
and capital letters in the same row .
mong samples in various concentratio.
indicate significance at p <0.
05 (Duncan post hoc tes.
The Indonesian Biomedical Journal.
Vol.
No.
August 2017, p.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179 Table 5.
IC50 ABTS-reducing activity of P.
niruri extract, rutin, and quercetin.
Samples niruri extract Rutin Quercetin Linear equation IC50 (AAg/m.
y = 30.
y = 28.
y = 30.
y = 2.
9161x - 0.
y = 2.
996x - 1.
y = 2.
9908x - 0.
y = 36.
y = 36.
y = 36.
Average IC50 (AAg/m.
53 A 0.
16 A 0.
17 A 0.
*The data was presented as mean A standard deviation of IC50 .
The most active DPPH scavenging activity was quercetin with the lowest value of IC50 Table 6.
FRAP activity of P.
niruri extract, rutin, and quercetin.
FRAP activity (M Fe(II)/ g extrac.
Concentration (AAg/m.
niruri extract 58 A 0.
Rutin Quercetin 95 A 1.
79 A 6.
92 A 0.
65 A 0.
50 A 1.
44 A 1.
85 A 0.
99 A 5.
53 A 1.
19 A 2.
86 A 4.
49 A 3.
58 A 1.
11 A 5.
95 A 2.
13 A 2.
28 A 0.
*The data was presented as mean A standard deviation.
Different superscript small letters in the same column .
mong concentrations of the sample.
and capital letters in the same row .
mong samples in various concentratio.
indicate significance at p <0.
05 (Duncan post hoc tes.
Discussion The qualitative phytochemical screening of P.
extract showed the presence of high content of saponin, the moderate content of phenol, flavonoid, tannin and low content of steroid.
However, triterpenoid, terpenoid, and alkaloid were undetected in the extract with the method used in this study (Table .
Based on other studies.
niruri contains tannins, terpenes, flavonoids, alkaloids, and saponins found in the leaves, stem, and roots of the plant.
Calixto, et al.
, reported that P.
niruri contained flavonoid quercetin, quercitrin, isoquercitrin, astragalin, rutin and .
Geraniin, repandusinic acid and corilagin are tannins found in P.
The alkaloid isolated from P.
niruri is a securinega-type alkaloid, .
The free radical scavenging activity of this plant could be due to the presence of various bioactive compounds such as alkaloids, flavonoids, and polyphenols has antioxidant capabilities.
The antioxidant assay of P.
niruri extract, rutin and quercetin revealed that quercetin possessed the greatest antioxidant activity, followed closely by P.
niruri extract in DPPH and ABTS assay (Table 2, .
In FRAP assay.
has the highest activity at the concentration of 25 AAg/mL, that comparable to quercetin.
The differences of antioxidant value among assays indicate that each assay determines different aspects in measuring antioxidant capacity and distinct radicals and mechanisms of reaction occurred.
Rutin exhibited the lowest activity among the samples in DPPH (Table 2, .
ABTS (Table 4, .
, and FRAP assay (Table .
based on the result, suggesting that rutin has the least antioxidant potential.
The high antioxidant activity of P.
niruri might be related to the numerous active compounds in the plant, including the phytochemicals flavonoids and DOI: 10.
18585/inabj.
Antioxidant of Phyllantus niruri Extract.
Rutin, and Quercetin(Rusmana D, et a.
Indones Biomed J.
: 84-90
This was in accordance with the current phytochemical screening results, which detected different phytochemical contents.
Based on the total phenol content assay in this study, it was also revealed that P.
contained high phenol content and the highest total phenol content was obtained using rutin as equivalent, with the value of TPC was 424.
29 AAg RE/mg extract.
The OH groups in phenols related to antioxidant activity, that can reduce of amount oxidation by transferring an H atom to chaincarrying radicals.
Mediani, et al.
, reported that P.
niruri contained a high amount of rutin, followed by gallic acid, quercetin.
The phenolic compound gallic acid and ellagic acid found in P.
niruri have been demonstrated to possess antioxidant activity and cancer chemopreventive abilities.
Rutin can reduce the risk of arteriosclerosis, anti-inflammatory and vasoactive properties, reduce coronary heart disease, and anticancer properties.
, .
In another study, quercetin has scavenging radicals activity and chelating transition metal ions that play a role in preventing oxidation of low-density lipoproteins (LDL) to against cancer, atherosclerosis, and .
In the present study, rutin has the lower antioxidant activity than quercetin.
These were consistent with Ahmeda, et al.
, who reported that quercetin has higher antioxidant activity than rutin in the DPPH assay due to the steric hindrance in rutin.
In another study conducted by Firuzi, et al.
, it was also demonstrated that quercetin had significantly higher antioxidant activity than rutin in FRAP assay, with FRAP value after 4 min of quercetin and rutin were 65.
0 and 21.
9 AAM, respectively.
Those results were assumed to be caused by the absence of 2,3-double bond or 3-OH in the C ring in the rutin structure, which were presumably important in determining antioxidant activity.
Among polyphenolic compounds, several factors could be responsible for increasing radical scavenging effectiveness.
Those factors are including ortho-dihydroxy structure in the B ring, 2,3-double bond in conjugation with a 4-oxo function in the C-ring, hydroxy groups in positions 3 and 5 in the A ring, or the angle between the rings in the compound Conclusion In summary.
niruri extract and quercetin have great potential as a natural antioxidant source.
References