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Anti-Oxidant activity in Natural Products

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Priyanka Singh
Priyanka Singh

ntioxidant natural products DPPH ABTS Free radicle FRAP Healthcare herbal Ayurvedic Immunity Natural polyphenol ORAC TRAPx ORAC assay antioxidant assay scavengers extraction technique natural antioxidant reducing agent

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Antioxidants Antioxidants are compounds capable to either delay or inhibit the oxidation processes which occur under the influence of atmospheric oxygen or reactive oxygen species In biological system, reactive oxygen species (ROS) and reactive nitrogen species (RNS),such as superoxide, hydroxyl, and nitric oxide radicals, can damage the DNA and lead to the oxidation of lipid and proteins in cells.
Anti-Oxidant activity in Natural Products
Anti-Oxidant activity in Natural Products
Mechanism of Anti-Oxidant The increment of intake of exogenous antioxidants would ameliorate the damage caused by oxidative stress through inhibiting the initiation or propagation of oxidative chain reaction acting as free radical scavengers quenchers of singlet oxygen reducing agents
The effective extraction (green extraction technologies) Many extraction factors play important roles in the extraction efficiency, such as Type and concentration of extraction solvent Extraction temperature Extraction time Extraction pH Proper assessment of antioxidants Chemical levels Cellular levels Crucial step for studying the natural antioxidants from plants
Extraction Methods of Antioxidants from Foods and Medicinal Plants
Green non-conventional methods Ultrasound-assisted extraction, Microwave-assisted extraction, Enzyme-assisted extraction, Pressurized liquid extraction, Supercritical fluid extraction, High hydrostatic pressure extraction, Pulsed electric field extraction and High voltage electrical discharges extraction.
Assessment Methods of Antioxidant Capacity - basis for ranking the antioxidant plants Chemical-based assays Cellular-based assays
Chemical-Based Assays- classified into 2 types acc. to mechanism (SET) single electron transfer Measure the ability of an antioxidant to transfer one electron to reduce target charged compounds, such as radicals and metal ions. a. Some assays are based on the ability to scavenge the stable free radicals (TEAC) Trolox equivalence antioxidant capacity DPPH assay FolinCiocalteu regent assay b. Some assays are based on the ability to reduce metal ions, such as (FRAP) ferric ion reducing antioxidant power (CUPRAC) cupric reducing antioxidant capacity
(HAT) hydrogen atom transfer Measure the ability of an antioxidant to quench free radicals by hydrogen donation, which are more relevant to the radical chain-breaking antioxidant capacity. HAT-based assays include (ORAC) oxygen radical absorbance capacity (TRAP) total radical trapping antioxidant parameter Inhibiting the oxidation of low-density lipoprotein (LDL).
Scavenging Free Radicals Assays ABTS (2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid) radical assay- The Trolox equivalent antioxidant capacity (TEAC) assay is widely applied to evaluate the antioxidant ability to scavenge the ABTS radical Potassium persulfate oxidize ABTS to generate its colored ABTS+ form (green color) Addition of antioxidants results in loss of the green color ABTS+ has a UV-vis absorption maximum at 734 nm. The decrease of absorbance can be monitored spectrophotometrically. The difference of the absorbance tested is plotted versus the antioxidants concentrations. The antioxidant capacity was expressed as Trolox equivalents.
ABTS radical assay- (2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid)
Merits of ABTS radical assay- ABTS+ could react rapidly with antioxidants, the assay possesses the merits of rapidity and simplicity ABTS+ is not influenced by ionic strength ABTS+ is soluable in both organic and aqueous solvents, so it can detect both hydrophilic and lipophilic antioxidant activities Demerits of ABTS radical assay- TEAC is inaccurate for slow reactions (duration of reaction is beyond 6 min).
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assay- Stable organic nitrogen radical The DPPH posses a deep purple color UV-vis absorption maximum at 515 nm The test compounds (antioxidants) reduce DPPH radical to DPPH-H and the solution color fades. The reducing ability can be assessed by measuring the decrease of its absorbance EC50 is the necessary amount of antioxidant to decrease the initial DPPH concentration by 50%
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assay-
Merits of DPPH radical assay- Easy, rapid and requires only a UV-vis spectrophotometer to test DPPH radical is commercially available and does not have to be generated before assay such as ABTS+. Demerits of DPPH radical assay- Application of DPPH assay is limited The linear reaction range of DPPH assay is narrow, only 23-fold Strong antioxidant activities in lipid peroxidation system may react slowly or may even be inert to DPPH
Reducing the Metal Ions Assays FRAPAssays The ferric-reducing antioxidant power (FRAP) assay measures directly the reducing capacity of antioxidants. The antioxidants can reduce a ferric tripyridyltriazine complex (Fe3+-TPTZ) to the ferrous complex (Fe2+-TPTZ) under pH 3.6 The ferrous complex (Fe2+-TPTZ) is blue ferrous form and has a UV-vis absorption maximum at 593 nm. FRAP value is positive related to the increase in absorbance
Merits of FRAP assay- Demerits of FRAP assay- FRAP cannot detect compounds that act by radical quenching (H transfer), particularly thiols and proteins. This results in a serious underestimation in serum sample assay is suitable for some antioxidants that complete the reaction rapidly within 4 to 6 min such as ascorbic acid and uric acid. Absorption of several dietary polyphenols in water and methanol slowly increased even after several hours, such as tannic acid, and caffeic acid
CUPRAC Assays similar to the FRAP method CUPRAC method is conducted by mixing Cu(II)-neocuproine (Nc) chelate with antioxidant solution. The absorbance of the color Cu(I)-chelate as a result of redox reaction is measured at 450 nm after 30 min Merits of CUPRAC Assays-reagent in this assay is useful at pH 7, which is at physiological pH (as opposed to the Folin and FRAP assays, which work at pH 10 and pH 3.6 applied for the determination of both hydrophilic and lipophilic antioxidants because the Cu(II)-Nc is soluble in both aqueous and organic environments (unlike Folin and DPPH) can measure the reducing power of thiol-type antioxidants, such as glutathione and nonprotein thiols
FolinCiocalteu Reagent (FCR) Assay method for quantitative determination phenolic compounds The mechanism of FolinCiocalteu method is electron transfer (ET) It involves reducibility of phenols in alkaline solution (pH = 10), which is capable of turning yellow molybdotungsto-phosphoric heteropolyanion reagent into the blue resultant molybdotungsto-phosphate These blue pigments have a maximum absorption in the 700760 nm range maximum absorption depends on the qualitative and/or quantitative composition of phenolic mixtures. The total phenols assay by FCR is simple & convenient,
Oxygen Radical Absorbance Capacity (ORAC) Assay In the basic assay,the peroxyl radical mixes with a fluorescent probe (FL; 30,60-dihydroxyspiro[isobenzofuran-1[3H],90[9H]-xanthen]-3- one), then form a nonfluorescent product, which can be quantitated easily by fluorescence When an antioxidant is added into the mixture, peroxyl radical induced oxidation is inhibited and the decay of FL is prevented. Antioxidant capacity is reflected by determining the decreased rate and amount of product formed over time
Total Radical Trapping Antioxidant Potential (TRAP) Assay The total radical trapping antioxidant potential (TRAP) assay measures the ability of antioxidants to suppress the oxidation progress of 2,20-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) or 2,20-azobis(2-amidinopropane) dihydrochloride (ABAP) [148,174]. The variation in the reaction progress is monitored fluorometrically (lex = 495 nm and lem = 575 nm).
Inhibiting the Oxidation of Low-Density Lipoprotein (LDL) Assay The autoxidation of linoleic acid or LDL is induced by Cu(II) or AAPH. The peroxidation of the lipid components is monitored at 234 nm by UV spectrometer for conjugated dienes. In the presence of a radical initiator, the reaction starts and the absorbance at 234 nm increases as the evidence of the accumulation of conjugated diene oxides. After the addition of antioxidants, the reaction rate slows down until the antioxidant is exhausted. In the period, the lag time is measured and used to evaluate antioxidant capacity.
Anti-Oxidant activity in Natural Products

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Anti-Oxidant activity in Natural Products

  • 1. P
  • 2. Antioxidants Antioxidants are compounds capable to either delay or inhibit the oxidation processes which occur under the influence of atmospheric oxygen or reactive oxygen species In biological system, reactive oxygen species (ROS) and reactive nitrogen species (RNS),such as superoxide, hydroxyl, and nitric oxide radicals, can damage the DNA and lead to the oxidation of lipid and proteins in cells.
  • 5. Mechanism of Anti-Oxidant The increment of intake of exogenous antioxidants would ameliorate the damage caused by oxidative stress through inhibiting the initiation or propagation of oxidative chain reaction acting as free radical scavengers quenchers of singlet oxygen reducing agents
  • 6. The effective extraction (green extraction technologies) Many extraction factors play important roles in the extraction efficiency, such as Type and concentration of extraction solvent Extraction temperature Extraction time Extraction pH Proper assessment of antioxidants Chemical levels Cellular levels Crucial step for studying the natural antioxidants from plants
  • 8. Green non-conventional methods Ultrasound-assisted extraction, Microwave-assisted extraction, Enzyme-assisted extraction, Pressurized liquid extraction, Supercritical fluid extraction, High hydrostatic pressure extraction, Pulsed electric field extraction and High voltage electrical discharges extraction.
  • 9. Assessment Methods of Antioxidant Capacity - basis for ranking the antioxidant plants Chemical-based assays Cellular-based assays
  • 10. Chemical-Based Assays- classified into 2 types acc. to mechanism (SET) single electron transfer Measure the ability of an antioxidant to transfer one electron to reduce target charged compounds, such as radicals and metal ions. a. Some assays are based on the ability to scavenge the stable free radicals (TEAC) Trolox equivalence antioxidant capacity DPPH assay FolinCiocalteu regent assay b. Some assays are based on the ability to reduce metal ions, such as (FRAP) ferric ion reducing antioxidant power (CUPRAC) cupric reducing antioxidant capacity
  • 11. (HAT) hydrogen atom transfer Measure the ability of an antioxidant to quench free radicals by hydrogen donation, which are more relevant to the radical chain-breaking antioxidant capacity. HAT-based assays include (ORAC) oxygen radical absorbance capacity (TRAP) total radical trapping antioxidant parameter Inhibiting the oxidation of low-density lipoprotein (LDL).
  • 12. Scavenging Free Radicals Assays ABTS (2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid) radical assay- The Trolox equivalent antioxidant capacity (TEAC) assay is widely applied to evaluate the antioxidant ability to scavenge the ABTS radical Potassium persulfate oxidize ABTS to generate its colored ABTS+ form (green color) Addition of antioxidants results in loss of the green color ABTS+ has a UV-vis absorption maximum at 734 nm. The decrease of absorbance can be monitored spectrophotometrically. The difference of the absorbance tested is plotted versus the antioxidants concentrations. The antioxidant capacity was expressed as Trolox equivalents.
  • 13. ABTS radical assay- (2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid)
  • 14. Merits of ABTS radical assay- ABTS+ could react rapidly with antioxidants, the assay possesses the merits of rapidity and simplicity ABTS+ is not influenced by ionic strength ABTS+ is soluable in both organic and aqueous solvents, so it can detect both hydrophilic and lipophilic antioxidant activities Demerits of ABTS radical assay- TEAC is inaccurate for slow reactions (duration of reaction is beyond 6 min).
  • 15. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assay- Stable organic nitrogen radical The DPPH posses a deep purple color UV-vis absorption maximum at 515 nm The test compounds (antioxidants) reduce DPPH radical to DPPH-H and the solution color fades. The reducing ability can be assessed by measuring the decrease of its absorbance EC50 is the necessary amount of antioxidant to decrease the initial DPPH concentration by 50%
  • 17. Merits of DPPH radical assay- Easy, rapid and requires only a UV-vis spectrophotometer to test DPPH radical is commercially available and does not have to be generated before assay such as ABTS+. Demerits of DPPH radical assay- Application of DPPH assay is limited The linear reaction range of DPPH assay is narrow, only 23-fold Strong antioxidant activities in lipid peroxidation system may react slowly or may even be inert to DPPH
  • 18. Reducing the Metal Ions Assays FRAPAssays The ferric-reducing antioxidant power (FRAP) assay measures directly the reducing capacity of antioxidants. The antioxidants can reduce a ferric tripyridyltriazine complex (Fe3+-TPTZ) to the ferrous complex (Fe2+-TPTZ) under pH 3.6 The ferrous complex (Fe2+-TPTZ) is blue ferrous form and has a UV-vis absorption maximum at 593 nm. FRAP value is positive related to the increase in absorbance
  • 19. Merits of FRAP assay- Demerits of FRAP assay- FRAP cannot detect compounds that act by radical quenching (H transfer), particularly thiols and proteins. This results in a serious underestimation in serum sample assay is suitable for some antioxidants that complete the reaction rapidly within 4 to 6 min such as ascorbic acid and uric acid. Absorption of several dietary polyphenols in water and methanol slowly increased even after several hours, such as tannic acid, and caffeic acid
  • 20. CUPRAC Assays similar to the FRAP method CUPRAC method is conducted by mixing Cu(II)-neocuproine (Nc) chelate with antioxidant solution. The absorbance of the color Cu(I)-chelate as a result of redox reaction is measured at 450 nm after 30 min Merits of CUPRAC Assays-reagent in this assay is useful at pH 7, which is at physiological pH (as opposed to the Folin and FRAP assays, which work at pH 10 and pH 3.6 applied for the determination of both hydrophilic and lipophilic antioxidants because the Cu(II)-Nc is soluble in both aqueous and organic environments (unlike Folin and DPPH) can measure the reducing power of thiol-type antioxidants, such as glutathione and nonprotein thiols
  • 21. FolinCiocalteu Reagent (FCR) Assay method for quantitative determination phenolic compounds The mechanism of FolinCiocalteu method is electron transfer (ET) It involves reducibility of phenols in alkaline solution (pH = 10), which is capable of turning yellow molybdotungsto-phosphoric heteropolyanion reagent into the blue resultant molybdotungsto-phosphate These blue pigments have a maximum absorption in the 700760 nm range maximum absorption depends on the qualitative and/or quantitative composition of phenolic mixtures. The total phenols assay by FCR is simple & convenient,
  • 22. Oxygen Radical Absorbance Capacity (ORAC) Assay In the basic assay,the peroxyl radical mixes with a fluorescent probe (FL; 30,60-dihydroxyspiro[isobenzofuran-1[3H],90[9H]-xanthen]-3- one), then form a nonfluorescent product, which can be quantitated easily by fluorescence When an antioxidant is added into the mixture, peroxyl radical induced oxidation is inhibited and the decay of FL is prevented. Antioxidant capacity is reflected by determining the decreased rate and amount of product formed over time
  • 23. Total Radical Trapping Antioxidant Potential (TRAP) Assay The total radical trapping antioxidant potential (TRAP) assay measures the ability of antioxidants to suppress the oxidation progress of 2,20-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) or 2,20-azobis(2-amidinopropane) dihydrochloride (ABAP) [148,174]. The variation in the reaction progress is monitored fluorometrically (lex = 495 nm and lem = 575 nm).
  • 24. Inhibiting the Oxidation of Low-Density Lipoprotein (LDL) Assay The autoxidation of linoleic acid or LDL is induced by Cu(II) or AAPH. The peroxidation of the lipid components is monitored at 234 nm by UV spectrometer for conjugated dienes. In the presence of a radical initiator, the reaction starts and the absorbance at 234 nm increases as the evidence of the accumulation of conjugated diene oxides. After the addition of antioxidants, the reaction rate slows down until the antioxidant is exhausted. In the period, the lag time is measured and used to evaluate antioxidant capacity.