Phytochemical Analysis, In Vitro Free Radical Scavenging, and LDL Protective Effects of Different Solvent Fractions of Calotropis procera (R.) Br. Root Bark Extract

In the present study


Introduction
Excessive oxidative stress plays a crucial role in cellular damage and apoptosis and is recognized as a major contributor to the pathogenesis of chronic diseases such as cancer [1], stroke [2], diabetes, cardiovascular diseases [3,4], atherosclerosis, chronic obstructive pulmonary disease, Alzheimer's, and other neurodegenerative diseases [5].It is thought that numerous malignancies arise due to the interplay of free radicals and DNA, leading to genetic alterations that impede the process of cell division [6].
Overproduction of ROS causes structural modifcations of cellular proteins and alters their functions, which leads to cellular dysfunction and disruption of essential cellular processes [7,8].A variety of mechanisms are involved in ROS-induced protein damage, including site-specifc amino acid modifcations, electric charge alteration, peptide chain fragmentation, aggregation of cross-linked products, enzyme inactivation, and proteolysis susceptibility [9,10].
Phenolics are one of the diverse arrays of phytochemicals occurring ubiquitously in food and medicinal plants as secondary metabolites.Te implication of reactive oxygen species in a wide variety of pathobiological manifestations and the benefcial role of polyphenols as potential natural antioxidants have been extensively studied and emphasized in previously published reports [11,12].Antioxidant phytochemicals, which are present in a variety of foods and medicinal plants, are crucial for the prevention and management of chronic diseases caused by oxidative stress.Tey are reported to have potent anti-infammatory, antioxidant, and free radical scavenging properties, which also form the basis for additional biological activities and therapeutic benefts such as protection against cancer, cardiovascular disease, diabetes mellitus, obesity, and neurodegenerative diseases [13,14].Tey either function by scavenging reactive oxygen species or by defending the body's endogenous antioxidative defense processes [15].It has been documented that the antioxidant activity of phenolic compounds is mainly due to their redox characteristics, hydrogendonating, singlet oxygen quenching, chain breaking, and metal chelating capabilities [16,17].
Tere is still a growing need to identify plant species that have antioxidant potential and therapeutic usefulness.Calotropis procera (Ait.)R. Br. is a xerophytic shrub, belonging to the Asclepiadaecae family and has a wide presence throughout Asia and Africa.Te signifcant therapeutic potential of the diferent parts of the plant has been reported.Traditionally, the latex is used to treat vertigo, alopecia, hair loss, toothache, intermittent fevers, joint infammation, and paralysis, while the powdered root is used to treat helminthiasis, bronchitis, asthma, leprosy, dermatitis, and elephantiasis.Te leaves are used to relieve joint discomfort and reduce swelling [18].
Terefore, the present investigation was aimed at phytochemical analysis and evaluation of in vitro antioxidant and LDL protective efcacy of C. procera (Ait.)R. Br. root bark methanol crude extract and its diferent solvent fractions.

Plant Collection and
Processing.Roots of C. procera (Ait.)R. Br. were collected from the bank of Bhadar River in Dhoraji (21 °45.2724′N, 70 °25.1586′E),Gujarat, India.Te taxonomic status of the plant was verifed by Dr. Rajesh Raviya, Professor of Botany, Department of Biology, MVM Science and Home Science College, Rajkot, India.Te roots were washed with water and the bark was peeled of using a knife and air dried for 10 days.Te dried plant material was pulverized into powder and stored in an air-tight container till further usage.

Extraction and Fractionation
Method.C. procera root bark (100 g) was thoroughly extracted with methanol in a Soxhlet apparatus for 72 h at a regulated temperature (40 °C).Te fnal product was fltered using Whatman flter paper (No. 42).Te resulting fltrate was concentrated at a low temperature in a water bath (40 °C) to obtain C. procera root bark methanol crude extract (CPRME).Te crude extract was then subjected to solvent-solvent fractionation following the Kupchan method modifed by VanWagenen et al. [25].Briefy, the dried crude extract (20 g) was subjected to solvent-solvent partitioning with solvents of increasing polarity: n-hexane, dichloromethane, ethyl acetate, and methanol (3 × 200 mL for each solvent type) in the darkness.All fractions were evaporated to dryness at a low temperature of 40 °C to yield hexane fraction (HF), dichloromethane fraction (DMF), ethyl acetate fraction (EAF), and methanol fraction (MF), respectively.At each 2 Journal of Food Biochemistry step of solvent fractionation, 40 mL of distilled water was added.Methanol insoluble residues were considered as an aqueous fraction (AF).Fractions were stored in an air-tight container until further use.

Preliminary Phytochemical
Investigation.CPRME and solvent fractions were subjected to qualitative phytochemical screening for the presence of various secondary metabolites [26].
2.5.Qualitative TLC Fingerprinting.Crude extract/fractions (10 mg/mL) were prepared and fltered through Whatman I flter paper.5.0 μL of extract/fractions were applied as a thin band on 10 cm × 8 cm Silica gel 60 F254 TLC plate (Merck, Germany).Te plate was developed in a glass chamber presaturated with toluene: ethyl acetate: glacial acetic acid (8 : 2 : 0.5) for 20 min.After visualization of fuorescent bands under UV light, the plate was sprayed with freshly prepared anisaldehyde-sulfuric acid reagent and placed in an oven at 110 °C for 5 min for the development of color bands.

Total Phenolic Content (TPC).
Te quantifcation of total phenolic content in the crude extract and fractions was conducted using the modifed Folin-Ciocalteu method as described by Wolfe and colleagues [27].An aliquot of the crude extract/fractions (1 mg/mL) was mixed with 2 mL Folin-Ciocalteu reagent (diluted 1 : 10 v/v with water) and 2 mL of sodium carbonate (Na 2 CO 3 , 75 g/L).Te reaction mixture was vortexed for 15 s and allowed to stand in darkness for 30 min at 25 °C for color development.A standard curve was plotted using diferent concentrations of tannic acid as a reference standard (10,20,30,40, and 500 μg/mL).Absorbance was then measured at 760 nm using a UV spectrophotometer.Total phenolic content was expressed in terms of tannic acid equivalent (TAE/g dry extract or fraction).

Total Flavonoid Content (TFC).
Te method described by Ordoñez et al. [28] was used to determine the total favonoid content.To 0.5 mL of crude extract/fractions (1 mg/mL), 1.5 mL of methanol, 100 μL of aluminum chloride (AlCl 3 , 10% w/v), 100 μL of 1 M potassium acetate, and 2.8 mL of distilled water were added.After 1.5 h of incubation at room temperature, the absorbance was measured at 420 nm.A standard curve was plotted by preparing diferent concentrations of quercetin in methanol as a reference standard (20,40,60,80, and 100 μg/mL) and total favonoid content were expressed in terms of mg of quercetin equivalent (QUE/g dry extract or fraction).

DPPH
where A 1 is the absorbance of the sample (extract/fractions/ standard), while A 0 is the absorbance of the control.
To this, 1 mL of 0.1% naphthylethylenediamine (NEDA) was added and allowed to stand for 30 min in the dark to complete the diazotization process.Te same process was carried out with ascorbic acid at 25, 50, 100, 150, and 200 μg/ mL concentrations.Te intensity of a pink chromophore was recorded at 540 nm against the corresponding blank solution with PBS in place of the sample.Te % of nitric oxide radical scavenging was calculated using the following equation: where A 0 is the absorbance of the control and A 1 is the absorbance of the crude extract/fraction/standard.
where A 0 is the absorbance of the control and A 1 is the absorbance of the crude extract/fraction/standard.

Hydroxyl (OH
where A 0 � absorbance without sample, A 1 � absorbance with sample, and A 2 � absorbance of sample omitting sodium salicylate. 2.12.Superoxide ( • O2 − ) Radical Scavenging Activity.Superoxide radical scavenging activity of crude extract/ fractions was performed according to the method by Beauchamp and Fridovich [33] with some modifcations.Various concentrations of crude extract/fractions (100, 200, 300, 600, and 1000 μg/mL) were added to the reaction mixture containing 100 μL EDTA (0.1 M), 200 μL sodium cyanide (NaCN, 0.0015%), 50 μL ribofavin (0.12 mM), 100 μL nitroblue tetrazolium (NBT, 1.5 mM), and phosphate bufer (67 mM, pH 7.8) keeping the total volume up to 3 mL.After 15 min of consistent illumination, the optical density of the mixture at 530 nm was measured.Ascorbic acid at 25, 50, 100, 200, and 300 μg/mL was used as a reference standard.A parallel blank in the identical conditions was run with distilled water in place of the sample in the reaction mixture.Te percentage inhibition was calculated using the following formula: where A 0 � absorbance without sample, A 1 � absorbance with sample, and A 2 � absorbance of sample omitting NBT.

Metal Ion Chelating Activity.
Te ability of the crude extract/fractions to chelate iron ions was estimated as per Gülçin method [34].Diferent concentrations of crude extract/fractions (100, 200, 300, 600, and 1000 μg/mL) and EDTA as standard (25,50,100,200, and 300 μg/mL) were added to 2.5 mL of 2 mM FeCl 3 .Te reaction was initiated by the addition of 0.2 mL of 5 mM ferrozine (fnal volume adjusted to 4 mL with methanol), mixed thoroughly, and allowed to stand at room temperature for 10 min.Te absorbance of the color produced was measured at 562 nm.Te percentage of inhibition was calculated using the following formula: where A 0 � absorbance of the control, containing FeCl 3 and ferrozine only, A 1 � absorbance in the presence of the tested samples, and A 2 � absorbance of the sample under identical conditions as A 1 with methanol instead of FeCl 3 solution.

Total Antioxidant Capacity (TAC).
Te TAC of crude extract/fractions was estimated as per the method reported by Prieto et al. [35] with some modifcations.An aliquot (0.5 mL) of crude extract/fractions (1 mg/mL) was mixed with 3 mL of the reaction mixture containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 1% ammonium molybdate.Te tubes were incubated at 95 °C for 10 min and the optical density was recorded at 695 nm using a spectrophotometer against blank after cooling at room temperature.
Te same approach was used to plot an ascorbic acid standard curve at various concentrations (25,50,100,200, and 300 μg/mL).Te results were expressed as mg ascorbic acid equivalents (AAE/g dry extract or fraction).

Reducing Power.
Te reducing power of crude extract/fractions was determined according to the method described by Oyaizu [36] with some modifcations.To 2.5 mL extracts/fractions of various concentrations (100, 200, 400, 800, and 1200 μg/mL) and ascorbic acid (25,50,100,200, and 300 μg/mL), 2.5 mL of phosphate bufer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (K 3 Fe(CN) 6 , 1%, w/v) were mixed.Te resultant mixture was incubated at 50 °C for 20 min.Te reaction was then stopped by adding 2.5 mL of 10% of TCA solution and then centrifuged at 3000 rpm for 10 min supernatant was collected.2.5 mL of supernatant, 2.5 mL of distilled water, and 0.5 mL of ferric chloride solution (0.1%, w/v) were mixed thoroughly.Te absorbance of the greenish-blue chromogen was measured at 700 nm.Higher absorbance of the reaction mixture indicated greater reducing power.
A parallel blank was run replacing the sample with 2.5 mL distilled water.

Lipid Peroxidation
2.16.1.Isolation of Human Serum LDL.LDL was isolated using a heparin-citrate bufer precipitation method developed by Wieland and Seidel [37].Te hyperlipidemic plasma was vortexed with 50 mL of heparin-citrate bufer (prepared by adding 5000 IU/L heparin to 100 mL 0.064 M trisodium citrate, pH 5.05 adjusted with 5 M HCl) and incubated for 10 minutes at room temperature.Te white precipitates were centrifuged at 3500 rpm at 4 °C and resuspended in 1 mL of phosphate-bufered saline (PBS, pH 7.4).LDL-C protein content was determined using the Lowry method modifed by Pomory using bovine serum albumin as standard [38].

Induction of LDL Oxidation.
In this study, LDL oxidation was performed according to the method described by Chumark et al. [39].An aliquot of LDL suspension (containing 200 μg LDL) in 10 mM phosphate bufered saline (PBS, pH 7.4) was incubated with 100 μL of diferent concentrations (25,50,100,200, 500, 1000, and 1500 μg/mL) of CPRME crude extract and its solvent fractions in a total volume of 1.5 mL for 30 min at 37 °C.LDL oxidation was initiated by adding 10 μL of freshly prepared 0.167 mM CuSO 4 to all tubes and incubating again for 6 h.At the end of the incubation period, oxidation kinetic was terminated by adding 10 μL EDTA (10 mM).

Measurement of Tiobarbituric Acid Reactive Substance (TBARS) in LDL.
Based on a method described by Okhawa et al. and modifed by Pulla and Lokesh [40], the extract/fractions were tested for their ability to inhibit CuSO 4 -induced LDL oxidation by measuring the amount of TBARS formed.Briefy, 0.5 mL of incubated LDL was mixed with 1 mL of KCl (1.15 M) and 2 mL of chilled thiobarbituric acid (TBA) reagent (0.25 M HCl, 15% trichloroacetic acid, 0.38% TBA, and 0.055% butylated hydroxy toluene).Te reaction mixtures were kept in a boiling water bath for 60 min at 100 °C and absorbance was measured at 570 nm.Te amount of TBARS was calculated using a molar extinction coefcient of 1.56 × 105 M −1 cm −1 and expressed as nmoles of TBARS/mg LDL protein.

GC-MS/MS Analysis.
Gas chromatography-mass spectrometry (GC-MS) analysis of EAF was performed at the Sophisticated Instrumentation Centre for Applied Research and Testing (SICART), Vallabh Vidyanagar, Anand, Gujarat.Analysis was performed using Autosystem XL with a turbo mass GC-MS spectrometer (Pekin Elmer, USA) coupled with a Quadrupole analyzer.Te GC-MS system was equipped with a PE-5MS column packed with 5% phenyl polysiloxane (30 m × 0.25 mm inner diameter).Helium (99.99%) was used as carrier gas at a fow rate of 1 mL/ min and a split ratio of 1 : 10.Temperature programming was applied (starting at 78 °C for 5 min; and increasing at 10 °C/ min up to 300 °C and held for 20 min).Te sample of EAF was prepared by accurately weighing 10 mg in 5 mL of methanol.Te solution was fltered through Whatman I flter paper, along with 2 g sodium sulfate to remove the sediments and traces of water in the fltrate and 1 μL of the solution was used for GC-MS analysis.Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 20-620 amu.To obtain the spectral data, separated chromatograms of various phytoconstituents were then subjected to mass fragmentation.Mass fragmentation was interpreted by comparing the spectral data with the database of the National Institute of Standards and Technology (NIST) and identifying compounds.

Statistical Analysis.
All tests were performed in triplicates (n � 3).Data are expressed as Mean ± SEM.Te statistical analysis was performed by one-way ANOVA followed by Dunnett's test using GraphPad Prism version 6.05 for Windows, GraphPad Software, San Diego, CA, USA.P < 0.05 was considered statistically signifcant.

Extraction and Fractionation
3.1.1.Preliminary Phytochemical Investigation.Table 1 shows the presence of secondary metabolites in CPRME crude extract and solvent fractions.Qualitative analysis revealed alkaloids, glycosides, triterpenoids, sterols, favonoids, and phenolic acids depending on the polarity of solvents used for fractionation and the chemical nature of the phytochemicals.Te presence of polyphenolic compounds in extract/fractions is also evident from diferent color bands obtained in the qualitative TLC fngerprinting (Figure 1).

Total Phenolic and Flavonoid Contents.
Te TPC and TFC of CPRME and its solvent fractions were measured (Figure 2).Te TPC and TFC were calculated using the regression equation of the standard curve plotted using tannic and quercetin as standard.Among the extract/fractions tested, EAF showed the highest amount of TPC (25.7 ± 3.12 mg TAE/g dry fraction), followed by DMF (19.05 ± 3.29 mg TAE/g dry fraction), CPRME (15.38 ± 2.21 mg TAE/g dry extract), and MF fraction (12.0 ± 2.37 mg TAE/g dry fraction).HF fraction showed the lowest amount of TPC (3.73 ± 0.98 mg TAE/g dry fraction) and TFC was not even detected in it.Te highest TFC was found in DMF fraction (13.69 ± 1.74 mg QUE/g dry fraction) followed by EAF (11.4 ± 1.88 mg QUE/g dry fraction), CPRME (10.36 ± 1.51 mg QUE/g dry extract), and MF fraction (9.09 ± 0.82 mg QUE/g dry fraction).Aqueous fraction (AF) showed the lowest amount of TPC (7.78 ± 1.43 mg TAE/ g dry fraction) and TFC (1.76 ± 0.63 mg QUE/g dry fraction) when compared with their counterparts.

Journal of Food Biochemistry
6 Journal of Food Biochemistry    and HF, showed iron chelation at slightly higher than 1000 μg/mL concentration (Table 2).EDTA, a reference standard, showed the lowest IC 50 values (150.03μg/mL).

Total Antioxidant Capacity.
Figure 4 shows the total antioxidant capacity (TAC) of solvent fractions of CPRME.Te total antioxidant activity is expressed as mg ascorbic acid equivalents (AAE).TAC was in the order of > DMF > CPRME > MF > AF > HF.Te TAC for EAF was found to be 94.14 ± 9.11 mg AAE, the highest among the fractions.Tis was followed by DMF (68.10 ± 8.78 mg AAE), CPRME (57.21 ± 5.66 mg AAE), MF (41.80 ± 4.69 mg AAE), AF (32.07 ± 7.87 mg AAE) and HF (16.06 ± 1.13 mg AAE). 5 depicts the reductive power of CPRME and its diferent solvent fractions.An increase in absorbance is suggestive of higher reducing power.Te results showed a concentration-dependent increase in the absorbance at 700 nm for extract fractions and the positive control ascorbic acid.Ascorbic acid showed a very powerful reducing ability.EAF containing a good amount of TPC and TFC was more powerful in reducing Fe 3+ to Fe 2 , followed by DMF, CPRME crude extract, MF, and AF.A negligible reducing activity was observed with HF.

LDL Protective Efects.
Since oxidized LDL (ox-LDL) has been implicated in the initiation and progression of atherosclerosis, hyperlipidemic agents with additional LDL protective efects would be more appreciated than the agents with only hyperlipidemic efects.For this reason, we also measured the protective efect of CPRME and its diferent solvent fractions on copper-induced LDL oxidation in the present study.Diferent concentrations of CPRME and its solvent fractions (25,50,100,200, 500, 1000, and 1500 μg/ml) were pre-incubated with LDL followed by CuSO 4 modifcation for 6 h (Figure 6).In this study, we have kept two additional groups: negative control (only native LDL without CuSO 4 and sample treatment) and positive control (native LDL with CuSO 4 only).Post 6 h of incubation of LDL with CuSO 4 , there was a signifcant increase (P < 0.001) of thiobarbituric acid reactive substances (TBARS) in the reaction mixture, suggesting copper-induced oxidative modifcation LDL when compared with normal control tubes.Te inhibitory efcacy of the CPRME (crude) and fractions on LDL modifcation is in the following order: EAF > DMF > HF > CPRME > MF > AF (P < 0.01 for AF, P < 0.001 for all other groups) when compared with positive control.EAF signifcantly  reduced the formation of TBARS in a dose-dependent manner when compared with positive control while DMF, HF, and CPRME required higher concentrations to produce the same efect observed with EAF.

Discussion
Antioxidants contribute to the removal of these oxidative products and slow down the process of oxidative modifcation and thereby preventing damage to the biological macromolecules such as cellular proteins, lipids and nucleic acids, and disruption of mitochondrial respiration [41].Polyphenols found in plants are secondary metabolites that have one or more hydroxyl groups attached to one or more aromatic rings.Several researchers have demonstrated that these plant polyphenols can serve as antioxidants to combat oxidative stress-induced diseases [42].In addition to polyphenolics, pentacyclic triterpenes, a group of secondary plant metabolites such as lupeol, α-amyrin, β-amyrin, and ursolic acid are also reported to have antioxidant properties and are valuable as antiinfammatory, antihypertensive, antiviral, antiangiogenic, antitumor, and antiangiogenic agents [24].Tese antioxidant compounds can scavenge radical oxygen species such as superoxide, hydroxyl, nitric oxide, and peroxides and consequently impede pathogenic oxidative processes of chronic diseases such as cardiovascular diseases, diabetes, neurodegenerative diseases, and cancer [1].Herbs have been regarded as efective antioxidants since ancient times.In the present study, total phenolic and favonoid contents were estimated in crude extract/ fractions.Te results indicate that polyphenolic compounds were accumulated in nonpolar and semipolar fractions.EAF showed the highest phenolic content while favonoid content was higher in DMF.Te results are in agreement with the previously reported studies [43,44].
For the evaluation of the free radical scavenging abilities of bioactive fractions of plant extracts and foods, the DPPH (1,1-diphenyl 1-2-picryl-hydrazyl) test has been widely utilized in phytomedicine.DPPH which is a stable nitrogencentered radical can accept hydrogen atoms from antioxidants to form a stable diamagnetic molecule [45].Terefore, when an antioxidant reacts with DPPH, the electron is paired of and the DPPH solution decolorizes.Tis reaction yields a stable product 1,1-diphenyl-2-picryl hydrazine by donating hydrogen atoms, resulting in a color change from purple to yellow which can be monitored as a decrease in absorbance at 517 nm [46][47][48].Te number of electrons taken up determines the antioxidant's ability to scavenge free radicals or bleach the color stoichiometrically [45].In the present study, the CPRME and subfractions were found to have varied amounts of phenolic and favonoid contents.Te results suggest that solvent fractions EAF and DMF followed by CPRME crude extract showed signifcantly higher DPPH + scavenging activity that positively correlated with their phenolic compounds such as favonoids, tannins, and phenolic acids.Te EAF contained the highest total phenolic content followed by DMF, whereas total favonoid content was higher in DMF than in EAF.Tese results are in accordance with the previously reported studies [43,44].Te hydroxyl groups (-OH) present in these polyphenolic compounds have the ability to donate hydrogen atoms to the DPPH radical, thereby neutralizing it.Te antioxidant activity of these compounds depends on the molecular structure, in particular on the number and position of hydroxyl groups and the nature of substitutions on the aromatic rings [49].Some favonoids such as rutin, isorhamnetin, and dihydroquercetin have been reported from C. procera root bark [50,51] and have efcient antioxidant activity [52][53][54].Terefore, the observed DPPH + 10 Journal of Food Biochemistry scavenging activity may probably be due to the hydrogendonating capability of the polyphenolic compounds present in EAF and DMF [55].
Nitric oxide (NO) is an essential bifaceted bioregulatory molecule with many physiological functions and pathological implications.Physiological efects include smooth

Journal of Food Biochemistry
Journal of Food Biochemistry muscle relaxation, blood pressure regulation, inhibition of platelet aggregation, neural signal transmission, and immune response [56].It has a very short half-life and may act as a highly unstable NO radical.Although NO does not interact with biological macromolecules directly, excessive superoxide anions in some chronic diseases react with NO to form peroxynitrite anion ( • ONOO − ), which prevent sodium transport across membranes, inhibit mitochondrial respiratory chain enzymes, and reduce cellular oxygen consumption.Tese anions even cause neuronal damage and DNA fragmentation and also participate in the pathogenesis of infammation [57,58].Te nitric oxide radical scavenging activity of CPRME crude extract and solvent fractions was measured by the Greiss method.NO • , spontaneously generated in the aqueous solution of sodium nitroprusside at physiological pH, could interact with oxygen to produce nitrite ions, causing diazotization of sulphanilamide.Te diazotized product undergoes coupling with naphthylethylenediamine dichloride forming an azo-dye, the intensity of which is measured at 550 nm.Phytochemicals with nitric oxide radical scavenging activity would compete with oxygen for nitric oxide thereby inhibiting the production of nitrite ions.Tis leads to a decrease in absorbance at 550 nm [58].In the present study, solvent fractions EAF and DMF showed considerable NO • scavenging efect followed by CPRME crude extract.Polyphenols are known to have antioxidant and free radical scavenging efcacy1.Due to the presence of aromatic structural elements, multiple hydroxyl groups, and a highly conjugated system, they can efectively scavenge reactive oxygen species (ROS).Tese free radicals can be neutralized by polyphenols by generating stable chemical complexes that halt subsequent detrimental processes.[59].Te free radicals nitric oxide and singlet oxygen (O-) participate in lipid peroxidation and are also implicated in the pathogenesis of chronic infammation [60].Polyphenols can inhibit the activity of inducible nitric oxide synthase (iNOs) or act as free radical scavengers to mitigate the propagation of infammation by NO [59].Te NO • scavenging property of EAF and DMF could be valuable in iNOS-generated excessive NO • radicals during chronic infammation in conditions such as atherosclerosis.H 2 O 2 is a slowly oxidizing, non-free radical species, but a substantial source of damaging hydroxyl radicals (OH • ).It can directly inactivate some enzymes by oxidation of essential thiol (-SH) groups.H 2 O 2 can cross cell membranes rapidly and participate in Fe 2+ and Cu 2+ -catalyzed Fenton reactions, giving rise to destructive hydroxyl radicals within the cells.Te resultant hydroxyl radicals may react with most of the biomolecules causing cell injury [61,62].It is, therefore, biologically advantageous for cells to regulate the amount of H 2 O 2 by decomposing it into oxygen and water.Te decomposition of H 2 O 2 by EAF and DMF was highest in comparison to other fractions.
Hydroxyl radical is regarded as the most reactive oxidant among ROS, causing lipid peroxidation to produce lipid hydroperoxides by abstracting hydrogen atoms from membrane polyunsaturated fatty acid and causing enormous biological damage in living systems [63,64].Lipid hydroperoxides are later decomposed to alkoxyl and peroxyl radicals and maintain the vicious cycle of oxidative cellular damage [65,66].In addition, it is a potent cytotoxic agent, capable of attacking the majority classes of biomacromolecules including proteins, DNA, and lipids and contributes to the pathological progression of many diseases such as atherosclerosis, diabetes, ischemic heart disease, cancer, Alzheimer's disease, and aging [67,68].Tus, it is very crucial to keep the hydroxyl radical at a minimum level for the protection of biological systems.Phenolic compounds with multiple hydroxyl groups exhibit high redox potential.Te antioxidant properties of phenolic compounds are signifcantly infuenced by the substituents on the phenyl ring and the conjugated carbon skeleton.[69].Due to the protective functions that antioxidants play in biological systems, investigating the radical scavenging and reducing capacities of antioxidants, especially those that are naturally present in plant sources is of signifcant interest.Te most common reaction mechanism proposed for scavenging hydroxyl (OH) radicals may include hydrogen atom transfer (HAT) by natural polyphenolic compounds.It has been reported that the most active site of • OH scavenging by polyphenols is the -OH group in the benzene ring by hydrogen atom transfer HAT mechanism [70].Te capability of solvent fractions EAF and DMF followed by CPRME crude extract to eliminate hydroxyl radicals could provide signifcant protection to biomolecules and impair pathobiological mechanisms implicated in chronic disorders.
Superoxide radicals although less reactive than hydroxyl radicals, which are continually produced during normal mitochondrial oxidative metabolism.Tey serve as precursors to the majority of ROS species and mediate oxidative chain events [71,72].However, excessively generated superoxide radicals are known to be damaging to biomolecules directly or indirectly by forming harmful H 2 O 2 , OH • , peroxynitrite anion ( • ONOO − ), or singlet oxygen during pathological events such as ischemic reperfusion injury [73].It was assumed that • O 2− could be the primary target of antioxidants against oxidative stress, indirectly decreasing the levels of other reactive species in the biological systems [71].Reduction of favins in the presence of light generates superoxide radicals which reduce nitroblue tetrazolium (NBT, yellow color), to blue-colored formazan which can be measured at 570 nm [33].Te present study suggests that DMF has the highest superoxide radical scavenging efect followed by EAF due to the presence of polyphenolic compounds; however, higher concentrations are required to quench the • O 2− radicals.It is suggested that plant polyphenols can mimic SOD enzyme action through the π-π interaction between superoxide and one polyphenol ring and is linked to oxidation of the superoxide radical, due to the transfer of its unpaired electron to an aromatic ring of the polyphenol and subsequent O 2 release during the reaction [74].
Trough the Fenton reaction, the ferrous ion (Fe 2+ ) can initiate lipid peroxidation by the breakdown of H 2 O 2 and decomposing lipid hydroperoxides into reactive hydroxyl-, peroxyl-, and alkoxyl-free radicals.Tis reaction gives Journal of Food Biochemistry momentum to the chain reaction of lipid peroxidation by abstracting hydrogen atoms from other vital molecules [75,76].Te metal chelating capacity of a compound eliminates excess catalyzing transition metals which is critical in the prevention of lipid peroxidation.It is well established that chelating agents decrease the redox potential by forming disulfde bonds with metal, thereby rendering the oxidized metal ion into a nonreactive form [77]. Ferrozine can make a complex with ferrous ions producing a purple color product.In the presence of chelating agents/extracts, the intensity of the purple color complex is reduced in a dose-dependent manner.Tis suggests that the plant components may either interfere in ferrozine-Fe 2+ complex formation or directly interact with Fe 2+ .Tus, the chelating efect of plant extracts as natural chelators can be determined by measuring the intensity of the purple color formed [78].A decrease in color intensity quantitatively correlates with higher metal chelating ability.Chelating agents that form bonds with metal are efective in reducing the redox potential and thereby stabilizing the oxidized form of the metal ions [79].Polyphenols are very efective metal chelators.Polyphenol-iron interactions (binding) have been proposed as a mechanism for the antioxidant behavior of the polyphenols.In a process known as autooxidation, polyphenol ligand complexes of Fe 2+ rapidly oxidize in the presence of oxygen to yield Fe 3+polyphenol complexes.Te binding of polyphenol ligands to Fe 2+ lowers the reduction potential of iron and accelerates the rate of iron oxidation.Te higher stability of the harder Fe 3+ metal ion interactions with the hard oxygen ligands of the polyphenol moieties, as well as the strong electron-donating properties of the oxygen ligands that stabilize the higher iron oxidation state, both contribute to this oxidation of Fe 2+ to Fe 3+ upon binding to polyphenol ligands [80].In this study, solvent fractions, notably EAF and MF have shown reasonable metal chelating activity, which may partly be due to interference of phytochemicals with Ferrozine-Fe 2+ complex formation, thereby establishing their role in the metal chelating activity.
Te phosphomolybdenum assay was used to assess the total antioxidant capacity of the crude extracts and fractions.Tis assay is based on the reduction of phosphomolybdanum ion from Mo (VI) to Mo (V) in the presence of an antioxidant with subsequent formation of a green phosphate/ MoV complex with maximal absorption at 695 nm under acidic conditions [81,82].In the present study, Te TAC for EAF was the highest among the fractions followed by DMF and CPRME (crude extract).Te observed efect could be attributed to their vast array of polyphenols and antioxidant triterpene compounds.
Te reducing power assay serves as a signifcant indicator of the overall antioxidant potential of plant extracts.Redox property, hydrogen donating ability, chain breaking potential during free radical generation, quenching transitionmetal ions, decomposition of hydroperoxides, inhibition of hydrogen extractions from biomolecules, radical scavenging, and reductive capacity have been proposed mechanisms contributing to the overall antioxidant potential of a plant [83,84].Te presence of various phenolic compounds, such as phenolic acids, favonoids, and tannins is typically linked to the reducing capabilities.Tey have a remarkable free radical chain-breaking ability by donating a hydrogen atom or reacting with certain precursors of peroxide, thus preventing peroxide formation [85,86].Te presence of reducing agents in plant extracts causes a reduction of the Fe 3+ -ferricyanide complex to the ferrous form leading to a color change from yellow to various shades of green and blue (Perl's Prussian blue) depending upon the number of reductants present.Te concentration of Fe 2+ in the system is directly proportional to the absorbance of the blue-green solution measured at 700 nm.Terefore, an increased absorbance is indicative of higher reducing power and the ability of a compound to donate electrons is suggestive of its antioxidant potency [87].In the present investigation, EAF containing a good amount of TPC and TFC were more powerful in reducing Fe 3+ to Fe 2+ , followed by DMF and CPRME (crude extract).
Lipid peroxidation is the ultimate result of oxidative damage to polyunsaturated fatty acids transforming membrane lipids into lipid peroxide radicals.Peroxidation of membrane lipids may disrupt membrane transport proteins, change the stability of ligand-binding sites on the membrane, and deactivate membrane-associated enzymes consequently, leading to cell death [88].In the present study, we also studied the efect of CPRME and solvent fractions on the copper-catalyzed human LDL oxidation, as assessed by the formation of a thiobarbituric acid reactive substance (TBARS).We found that CPRME crude extract, EAF, and DMF signifcantly counteracted the formation of TBARS.It is believed that the copper ion interacts with lipoprotein and degrades lipid hydroperoxides through a Haber-Weiss type reaction pathway.Tis phenomenon results in the formation of OH • , RO • , and ROO • radicals by abstracting a hydrogen atom from a fatty acid side chain at, or near the lipoprotein surface [89][90][91].Te results obtained from the present study indicate that CPRME and their solvent fractions are capable of afecting the rate of LDL oxidation at the end of 6 hr incubation.Tis could be due to the ability of the extracts to chelate or interfere with copper, thereby inactivating the redox mechanism and free radical generation in the assay system [92].We observed that the ethyl acetate fraction showed maximum inhibition of LDL oxidation.Tis could probably be due to the presence of moderately nonpolar components of EAF which may specifcally interact with some lipogenic radicals generated during copper-catalyzed oxidative processes within the hydrophobic core of LDL [93].Terefore, in the present investigation, the LDL protective efect of CPRME (crude) and its solvent fractions, specifcally EAF, DMF, and HF could also be attributed to the stabilization of the outer layer of LDL phospholipids.According to Brown et al., lipophilic phenolic compounds can be localized to the surface of phospholipid bilayers, which can protect against free radical attacks and perhaps sequester metal ions [94].On the other hand, hydrophilic antioxidants could not optimally access the lipid moiety of LDL and would thus be less efective in countering lipophilic radicals.Tese arguments on solubility and partitioning behavior of antioxidants suitably support the observed LDL 14 Journal of Food Biochemistry protective efect produced by EAF, DMF, and HF owing to their polar and nonpolar phytoconstituents having radical scavenging and transitional metal ion chelating actions.Tis efect could be useful in delaying atheromatous plaque development.

Conclusion
Te antioxidant potential of diferent solvent fractions of C. procera root bark methanol extract was evaluated using diferent radical systems.In this radical scavenging mechanism, polyphenols sacrifcially reduce ROS/RNS, such as • OH, • O 2 − , NO • , or OONO− after generation, preventing damage to biomolecules, or formation of more reactive ROS.Te present study confrms the free radical scavenging activity of crude extract and solvent fractions of C. procera root bark, notably EAF and DMF.Te observed antioxidant properties may be attributable to various secondary metabolites as these fractions contain high polyphenolic and pentacyclic triterpene compounds which correlate with their good antioxidant efcacy.Te presence of antioxidant pentacyclic triterpenes such as lupeol, α-amyrin, β-amyrin, and ursolic acid in EAF was characterized using GC-MS/MS approach.Te total phenolic content was highest in EAF followed by DMF whereas total favonoid content was highest in DMF.Te EAF produced signifcant DPPH + , NO • , H 2 O 2 , and OH • scavenging activity.Apart from that, DMF was more efective in • O 2 − scavenging activity while the MF showed the highest metal chelating activity.However, the EAF showed the highest TAC, probably due to the presence of antioxidants pentacyclic triterpenes such as lupeol and amyrins in addition to the polyphenolic compounds.In vitro antioxidant studies are relevant in exploring the potential health benefts of plant extracts or their solvent fractions.Tere is a growing interest within the community in using plantbased antioxidant therapies as complementary and alternative treatments, even in chronic diseases.Te current study demonstrates that the phenolic acids, favonoids, and pentacyclic triterpenes of EAF of C. Procera root bark methanol extract can aford the protection against free radicals-induced damage in many chronic health conditions and could be valuable in the prevention and treatment of many chronic diseases such as hyperlipidemia and atherosclerosis.Further studies are required to explore its therapeutic potential.

Figure 7
shows the gas chromatogram of EAF.Te gas chromatography-mass spectrometry (GC-MS) analysis of EAF revealed the presence of important secondary metabolites which were identifed by comparing their mass spectral fragmentation patterns with those of known compounds listed in the National Institute of Standards and Technology (NIST) library.

Table 1 :
Extraction of the yield of CPRME and its solvent fractions.

Table 2 :
24 50 values of CMRME and its solvent fractions on diferent in vitro radical systems tested.Te scavenging activity of CPRME and its solvent fractions against nitric oxide released by sodium nitroprusside was investigated by the Griess reagent method (Figure3(b)).In this study, the NO scavenging capacities of samples and the positive control increased in a dose-dependent manner.Te order of efcacy was observed to be EAF > DMF > CPRME > MF > AF > HF in a dose-dependent manner.A % inhibition shown by EAF was highest at the dose of 600 μg/ml (75.39 ± 3.214%) while that of CPRME, DMF, MF, AF, and HF was found to be 64.24±5.16, 67.32 ± 5.44, 55.19 ± 3.22, and 36.57± 3.52%, respectively, in decreasing order.Tese results were further consolidated by IC 50 analysis data (Table 2), showing the EAF fraction with the highest NO scavenging ability (IC 50 � 317.46 μg/mL) among the fractions.Other fractions, such as MF (IC 50 � 665.14 μg/mL) and AF (IC 50 � 887.41 μg/mL), required a higher concentration to scavenge H 2 O 2 , whereas the HF fraction could only provide 50% inhibition at a concentration of 2245.78 μg/mL (Table 2).Except for HF, all fractions displayed reasonable H 2 O 2 scavenging activity, though lower than the reference drug ascorbic acid (IC 50 � 57.23 μg/mL).

8
Journal of Food BiochemistryEAF was the highest among the fraction but lower than the reference drug ascorbic acid (IC 50 � 57.23 μg/mL).

Table 3 :
GC-MS analysis of EAF depicting retention time (RT), peak area, and compounds identifed.