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An#oxidant Effect of Water and Ethanol (80%) Extracts of Andrographis paniculata (Burm. f.)
Nees. leaves
Presented by Sekar Galuh (Indonesia)
Supervised by
Assoc. Prof. Dr. Jamia Azdina Jamal
Kuala Lumpur, 18th August 2016
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INTRODUCTION Andrographis paniculata Ness. (King of BiHer/Brotowali (indo)/Hempedu Bumi (malay)
• Major compounds : • Diterpenoid
(Andrographolide) • Flavonoid • Polyphenols
(Chao and Lin, 2010)
“Compounds in plant extracts, herbs, and syntheVc showed the an#oxidant ac#vity” (Brewer, 2011)
“Flavonoid showed many biological acVviVes, such as anVallergenic and anVinflammatory. An#oxidant capacity of flavonoid in vitro has been studied.” (PieHa, 2000)
“Polyphenols, as secondary metabolites of plants, is involved against ultraviolet radia#on” (Pandey and Rizvi, 2009)
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Objective
To quanVfy Phenolic and Flavonoid contents in water extract and ethanol 80% extract of Andrographis paniculata leaves
The aim of this research are
To evaluate anVoxidant acVvity of water extract and ethanol 80% extract of Andrographis paniculata leaves
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Methodology
Water and Ethanol 80% Extracts
QuanVfy Phenolic and Flavonoid Contents
Evaluate AnVoxidant AcVviVes
Total Phenolic Content (TPC)
Total Flavonoid Content (TFC)
Ferric Reducing AnVoxidant Power
(FRAP)
Diphenyl-‐picrylhidrazil
(DPPH)
Analysis (using Microsod Excel)
mg Gallic Acid Equivalent/g
mg QuerceVn Equivalent/g
mg Trolox Equivalent/g RSA; AAI; IC50
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Methodology Total Phenolic Assays (TPC) (ISO 14502,2005)
20 μL sample (1mg/mL)/
standard (Gallic acid)/DMSO
100 μL Folin-‐
Ciocalteu (10%)
IncubaVon 5’
96-‐well plates 80 μL Na2CO3 (75%)
Slightly shaken
IncubaVon 30min (darkness at RT)
Absorbance read at 735nm using microplate reader
Total Flavonoid Assays (TFC) (Yang et al, 2011) 100 μL sample/
standard (QuerceVn)/
DMSO
100 μL AlCl3 (2%)
IncubaVon 15’
96-‐well plates
Absorbance read at 435nm using microplate reader
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Methodology Ferric Reducing An#oxidant Power Ac#vity (FRAP) (Benzie & Strain, 1996; Yang et al, 2011)
20 μL sample (8-‐0.0156mg/mL)/standard
(Trolox; Ascorbic Acid)/
DMSO
100 μL FRAP reagent
shake 30sec
96-‐well plates IncubaVon 4 min 37°C
Absorbance read at 593 nm using microplate reader
Diphenyl-‐picrylhidrazyl Assay (DPPH) (Zongo et al, 2010)
100 μL sample (8-‐0.0156mg/mL)/standard (Ascorbic Acid)/
DMSO
100 μL DPPH SoluVon reagent
IncubaVon 15min at RT (dark room)
96-‐well plates
Absorbance read at 540 nm using microplate reader
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Results
y = 0.0049x + 0.0338 R² = 0.9955
0.0000
0.1000
0.2000
0.3000
0.4000
0.5000
0.6000
0 20 40 60 80 100
Absorban
ce
Concentra#on (ug/mL)
Standard Curve TPC
Series1
Linear (Series1)
No. Type of Extracts Concentra#on of Phenolic (μg/mL)
Total Phenolic Contents (mg GAE/g)
1. Water extract 12.4097 1979.2175 ± 239.19
2. Ethanol (80%) extract 19.9066 3132.4274 ± 72.46
TOTAL PHENOLIC CONTENT (TPC)
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Results
y = 0.0208x -‐ 0.1725 R² = 0.99284
0.0000
0.5000
1.0000
1.5000
2.0000
2.5000
0 20 40 60 80 100
Absorban
ce
Concentra#on (ug/mL)
Standard Curve TFC
Series1
Linear (Series1)
TOTAL FLAVONOID CONTENT (TFC)
No. Type of Extracts Concentra#on of Flavonoid (μg/mL)
Total Flavonoid Contents (mg QE/g)
1. Water extract 13.8880 1269.4681 ± 50.36
2. Ethanol (80%) extract 25.0878 7895.4420 ± 44.09
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Results FERRIC REDUCING ANTIOXIDANT ASSAYS (FRAP)
No. Serial Dilu#on (mg/mL)
Equivalent Trolox (mg TE/g)
Water extract Ethanol (80%) Extract
1. 0.5 3293.0087 3686.2979
2. 1 8795.7385 7259.0896
3. 2 15174.6569 12936.0615
4. 3 33478.3691 25521.3156
5. 8 63305.2887 49297.8875
y = 0.0106x -‐ 0.0459 R² = 0.99987
0.0000
0.2000
0.4000
0.6000
0.8000
1.0000
1.2000
0 50 100 150
Absorban
ce
Concentra#on (ug/mL)
Standard Curve Trolox
Series1
Linear (Series1)
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Results DIPHENYL-PICRYLHIDRAZYL (DPPH) ASSAY
No Serial Dilu#on (mg/mL)
Radical Scavenging Ac#vity (RSA)
An#oxidant Ac#vity Index (AAI)
IC50
Water extract
Ethanolic Extract
Water extract
Ethanolic Extract
Water extract
Ethanolic Extract
1. 0.0625 44.9251 45.3432
34.13481 40.04599 2.9287 ±0.08
2.4967 ±0.013
2. 0.125 46.1005 45.5141
3. 0.25 46.1400 46.2083
4. 0.5 46.4871 46.1321
5. 1 47.7020 46.9420
6. 2 49.5138 49.7952
7. 3 53.0980 52.7456
8. 8 55.8564 59.9350
Standard Curve Ascorbic Acid y = -‐0.0044x + 1.0858 R² = 0.99841
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Discussions
• Ethanol (80%) extract contains more phenolic and flavonoid compounds than water extract
• Its an#oxidant ac#vity is also higher than water extract • There is a linear relaVonship between natural compound and anVoxidant acVvity
• The higher phenolic and flavonoid exhibited in plants, the higher its anVoxidant acVvity
• Based on (Qader, 2011), Aqueos extract of Andrographis paniculata has more phenolic and higher anVoxidant capacity
• They might use different solvent • Phenolic and Flavonoid more soluble in ethanol because its polarity lower than water
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Conclusions
Ethanol (80%) extract of Andrographis paniculata leaves contains higher phenolic and flavonoid
Ethanol (80%) extract of Andrographis paniculata leaves has stronger anVoxidant acVvity
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Acknowledgement
1. Eldiza Puji Rahmi 2. Ms. KarVniwaV Muhammad 3. Nor-‐Ashila Aladdin 4. Zalita Yaacob 5. Faculty of Pharmacy UKM 6. SEP CommiHee MyPSA
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References
Benzie, I.F.F. & Strain, J.J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “anVoxidant power”: the FRAP assay. AnalyVcal Biochemistry 239: 70-‐76. Brewer, MS., 2011, Natural AnVoxidants: Sources, Compounds, Mechanisms of AcVon, and PotenVal ApplicaVons, Comprehensive Reviews in Food Science and Food Safety, 10 (4):221-‐247 Chao, Wen-‐Wan., Lin, Bi-‐Fong., 2010, IsolaVon and idenVficaVon of bioacVve compounds in Andrographis paniculata (Chuanxinlian), Chinese Medicine, 5:17 Pandey KB., Rizvi SI., 2009, Plant polyphenols as dietary anVoxidants in human health and disease, Oxid Med Cell Longev, 2(5): 270-‐278 PieHa, PG., 2000, Flavonoid as AnVoxidants, Journal Natural Products, 63(7):1035-‐42 Qader, SW., Abdulla, MA., Chua, LS., Najim, N., Zain, MM., Hamdan, S., 2011, AnVoxidant, Total Phenolic Content and Cytotoxicity EvaluaVon of Selected Malaysian Plants, Molecule, 16:3433-‐3443 Yang, H., Dong, Y., Du, H., Shi, H., Peng, Y. & Li, X. 2011. AnVoxidant compounds from propolis collected in Anhui, China. Molecules 16: 3444-‐3455. Zongo, C., Savadogo, A., OuaHara, L., Bassole, I.H.N., OuaHara, C.A.T., 2010. Polyphenols content, anVoxidant, and anVmicrobial acVviVes of Ampelocissus granVi (Baker) Planch. (Vitaceae): a medicinal plant from Burkina Faso. InternaVonal Journal of Pharmacology 6(6), 880-‐887.
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