Can We Check Antioxident Activity of Flavonides Directly From Recombenant Bacterila Culture?

• Research article
• Open Access
• Published: 22 May 2012

Assessment of flavonoids contents and in vitro antioxidant activity of Launaea procumbens

Chemistry Central Periodical book 6, Article number:43 (2012) Cite this commodity

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Abstract

Background

Launaea procumbens (LP) has been used equally a food supplement in Pakistan. In this written report methanolic crude excerpt (LPME) of the whole plant and its different fractions; n-hexane (LPHE); ethyl acetate (LPEE) and chloroform (LPCE) were studied for the determination of full flavonoid and phenolics contents along with multifaceted in vitro scavenging assays.

Results

Considerable corporeality of flavonoid and phenolics contents were found in all the fractions. Methanol and chloroform fraction exhibited efficient scavenging of DPPH·, ABTS·+, ·OH, superoxide, lipid peroxide and nitric oxide free radicals. Significant correlation was found between DPPH·, ABTS·+, superoxide radical, β-carotene bleaching restraint and phosphomolybdenum assay with total flavonoids and phenolics contents. High performance chromatography (HPLC) of LPME revealed the presence of vitexin, orientin, rutin, hyperoside, catechin and myricetin.

Determination

These results reveal the presence of bioactive compounds in LPME, which might exist contributed towards the various in vitro scavenging.

Background

Reactive oxygen species (ROS) are generated in the normal metabolism of living organisms, and besides of their useful role in point transduction; they are also involved in the dispersion of several degenerative diseases similar malignant tumors, rheumatic joint inflammation, cataracts, Parkinson'due south and Alzheimer'due south disease, hypertension, diabetes, oxidative stress, tissue damages and atherosclerosis [1]. To protect the body from such effects; in add-on to antioxidant enzymatic system, there are non-enzymatic biomolecules and proteins in living organisms, which deed as antioxidant and free radical scavengers. Yet, nutrient supplementation containing ascorbates, carotenoids, tocopherols, flavonoids and phenols play a significant office in this matter [2, three]. These bioactive natural compounds scavenge the reactive oxygen species and prevent complimentary radicals to cause deterioration. They have the aptitude to scavenge oxygen-nitrogen derived free radicals past altruistic hydrogen atom or an electron, chelating metal catalysts, activating antioxidant enzymes and inhibiting oxidizes [iv–6]. Based on such a type of incredible results, interest in exploration of bioactive compounds extracted from medicinal plants was increased in recent years to replace the use of synthetic drugs, which were restricted due to side effects. On the other hand, polyphenol, used as natural antioxidants, are gaining importance, due to their health benefits for humans, decreasing the risk of cardiovascular and degenerative diseases by reduction of oxidative stress and counteraction of macromolecular oxidation [7, 8].

Medicinal plants are as well in high demand for application of functional food or biopharmaceuticals because of consumer preferences. Launaea procumbens (LP) is one of the important medicinal plants widely distributed in waste product places, vacant lots and in cultivated fields throughout Pakistan. Ayurvedic and herbal medicine prepared from this establish promote cocky healing, skilful health and longevity, as well as used every bit a nutrient ingredient [9]. Traditionally, it has been used in the handling of kidney disorders similar painful urination, gonorrhea, and sexual diseases [10]. Chemical label showed that LP is composed of salicylic acrid, vanillic acid, synergic acid, 2-methylresercinol and gallic acid [11]. These compounds have spasmogenic, cardiovascular, anticarcinogenic, antiinflammatory, and antioxidant backdrop to scavenge reactive oxygen species [12]. The present study was arranged to screen the various fractions of LP for the conclusion of total flavonoids and phenoilc consents, and to evaluate its antioxidant potential through scavenging of diverse free radicals.

Results

Phytochemical characterization

Full phenolics and flavonoids contents

Table 1 shows the presence of phenolics and flavonoids contents in diverse fractions of LP. The LPME possessed the highest total phenolics contents (432.eight ± 2.93) mg GAE/g while n-hexane comprised of lowest total phenolics content (188.3 ± 2.1) mg GAE/g extract. Maximum full flavonoid contents were recorded in LPME (13.98 ± 0.87) while the lowest concentration was present in LPHE (four.43 ± 0.45) mg equivalent rutin/g of dry out fraction. The extraction yield of these samples varied from four.43 ± 0.45% to be 16.28 ± 0.27% with a descending order of methanol > chloroform > ethyl acetate > n-hexane fraction. Methanol and chloroform fractions resulted in the highest amount of total extractable compounds, whereas the extraction yield with ethyl acetate and n-hexane was significantly less (P < 0.01) equally compared to methanol and chloroform fraction.

Table 1 Total phenolic content in different extracts of Launaea procumbens

Full size table

HPLC quantification of flavonoids

The HPLC-UV chromatogram revealed the presence of six polyphenolic compounds, including kaempferol, orientin, rutin, hyperuside, myricetin and quercetin. The investigated compounds in the methanolic extracts were quantified past integration of the peak-areas at 220 nm using an external scale method. Scale curves were constructed for each standard compound. Least-squares linear regression was used to determine the scale parameters for each of standards. The linearity of all calibration curves was adamant past calculating the correlation coefficients. In that location were some modest peaks, which could non exist identified; still, based on their chromatographic behaviors and UV spectra, their chemical grade may represent to unknown flavonoids compounds equally presented in Effigy 1. The Tabular array two revealed that LPME possessed highest quantity of myricetin (1.237 ± 0.04) while hyperuside (0.335 ± 0.06) are in low concentration.

Effigy 1

HPLC fingerprints obtained by methanolic excerpt of LPME Column: C18 20RBAX ECLIPSE, XDB-C18, (5 μm; iv.six × 150 mm, Agilent USA) eluted with mixtures trifluoroacetic acid and acetonitrile indicated the presence of 6 compounds 1.; (kaempferol), ii.; (orientin), 3.; (rutin), four.; (hyperuside), 5.; (quercetin) and half dozen.; (myricetin).

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Tabular array 2 HPLC quantification of methanolic extract of Launaea procumbens

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In vitroantioxidant assays

DPPH (1, i-diphenyl-two-picryl-hydrazyl) radical scavenging activity

DPPH is a stable free radical, which has been widely used in phytomedicine for the cess of scavenging activities of bioactive fractions. The scavenging activities of diverse fractions of LP extracts were determined using free radicals of 1, i-diphenyl i-ii-picryl-hydrazyl (DPPH) (Figure two and Table 3). Results showed that LPME (IC50 2.six ± 0.004 μg/ml) possessed the highest antioxidant activity as compared to other fractions while LPHE had the everyman scavenging upshot (IC50 nineteen ± 0.04 μg/ml). The DPPH radical scavenging activities of the LPME were even less (P < 0.01) than those of ascorbic acid.

Figure 2

DPPH radical scavenging activity of dissimilar extracts from the methanol extract of 50. procumbens by dissimilar solvents at different concentrations. Each value represents a Mean ± SD (north = three) LPHE; LPEE; LPCE; LPME; ascorbic acid.

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Table 3 IC ₅₀ of different extracts of Launaea procumbens for various antioxidant systems

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ABTS radical cation assay

Scavenging capacities of diverse fractions of LP and ascorbic acid were assessed using ABTS (2, 2 azobis-(3-ethylbenzothiozoline-half dozen-sulphonic acid) radical cation. Diverse fractions were plant considerably different in their ABTS radical cation scavenging activities. The ABTS radical scavenging action orders of diverse fractions of LP are; LPME (50.2 ± 1.7 μg/ml) > LPCE (88.five ± iii.8 μg/ml) > LPEE (104.3 ± 6.ix μg/ml) > and LPHE (112.1 ± vii.ii μg/ml) respectively (Tabular array 3). The results showed that LPME possessed significantly higher ABTS radical scavenging activity (P < 0.01) as compared to ascorbic acid (57.4 ± 3.1 μg/ml).

Phosphomolybdenum assay

The bones principle to assess the antioxidant capacity through phosphomolybdenum assay includes the reduction of Mo (VI) to Mo (Five) past the plant extract possessing antioxidant compounds. In the nowadays study addition of the various fractions of LP showed that LPME (IC50 64.27 ± two.1 μg/ml) was more constructive in reduction of Mo (VI) to Mo (Five) while the lowest furnishings were shown by LPHE (123 ± iii.09 μg/ml). The reduction of Mo (VI) to Mo (V) by assistants of reference chemicals; ascorbic acid (IC50 72.three ± ii.2 μg/ml) (Table 3), suggested the presence of constructive antioxidants in various fractions of LP.

Antioxidant activity determined by β-carotene bleaching method

The antioxidant potential of the various fractions of LP was arranged for screening through β- carotene bleaching method (Table 3). The absorbance of β-carotene was found to be decreased in the presence of 50–250 μg/ml of the various fractions or ascorbic acid. Various fractions of LP effective inhibited the oxidation of linoleic acid and subsequent bleaching of β-carotene. Amidst the various fractions, LPME (IC50 51.iv ± 2.4 μg/ml) showed greater inhibitory activity (P < 0.01) of β-carotene than other fractions and ascorbic acrid (IC50 54.seven ± three.half dozen μg/ml). The results suggested that various fractions possessed effective antioxidant constituents.

Superoxide radical scavenging activity

Oxidation is life, just except of so many necessary processes of life, during normal metabolism of oxygen, various free radicals too as superoxide are produced continuously. The loftier level of this superoxide radical is known to be harmful to cellular ingredients as, contributing to tissue damage and diverse diseases. The scavenging of the various fractions of LP extracts on superoxide radicals are shown in Figure 3 and Table 3. Scavenging for super oxide radicals exhibited by LPME (IC50 70.3 ± 2.43 μg/ml) was comparatively like to ascorbic acrid.

Figure 3

Super oxide radical scavenging activeness of different extracts from the methanol extract of 50. procumbens by dissimilar solvents at unlike concentrations. Each value represents a Hateful ± SD (northward = iii) LPHE; LPEE; LPCE; LPME; ascorbic acid.

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Hydroxyl radical scavenging

Amongst the oxygen radicals, hydroxyl radical is the most reactive and induces astringent damage to adjacent biomolecules such as protein, Dna and lipids; cause'due south lipids peroxidation. Table 3 shows the hydroxyl radical scavenging of the various fractions and ascorbic acid. LPME, LPEE, LPCE and LPHE scavenged hydroxyl radicals by IC50; 51.2 ± 1.4 μg/ml, 56.4 ± 2.0 μg/ml, 75.iii ± two.23 μg/ml and 92.5 ± 0.56 μg/ml, respectively. The scavenging affects of ascorbic acrid (IC50; 92.5 ± 2.56 μg/ml) were significantly lower confronting the various fractions of LP.

Hydrogen peroxide-scavenging

Hydrogen peroxide is nonreactive, simply its high concentrations are toxic to living cells, and changed into free radical called hydroxyl radicals; therefore, the scavenging affects of diverse fractions are evaluated confronting this free radical (Tabular array 3). The hydroxyl free radical in the cells can easily cross prison cell membranes and react with near biomolecules causes tissue damage, cancer and jail cell decease. Thus, removal of hydroxyl free radical is necessary in to protect life. Scavenging touch on of various fractions with ascorbic acid showed that LPME (IC50; 63.iv ± 3.65 μg/ml) had (P < 0.01) highest hydroxyl radical scavenging bear upon and was most potent than ascorbic acid (IC50 76.3 ± 2.15 μg/ml) respectively.

Chelating on Fe2+

Chelation of iron plays the main role for assessing antioxidant potential of medicinal plants. The reducing power of various fractions to reduce iron ion Fe (III) into Iron (II) is shown in Table iii. Various fractions of LP showed an ability to chelate atomic number 26 (II) ions in a dose-dependent style. LPCE and LPME chelated iron ion (IC50; 69.5 ± 3.0 μg/ml; IC50; 63.6 ± i.ane μg/ml), all the same, LPEE and LPHE chelate iron (Ii) ions (IC50; 74.1 ± three.06 μg/ml, IC50; 92.five ± 3.25 μg/ml), as confronting the iron chelating for ascorbic acrid was IC50; 65.0 ± 2.1 μg/ml.

Lipids peroxidation analysis

Egg yolk lipids undergo rapid nonenzymatic peroxidation when hatched in the presence of ferrous sulfate. Lipids peroxides are likely involved in many pathological events, including inflammation, metabolic disorders, oxidative stress and cellular aging. The affects of various fractions of LP, ascorbic acids on nonenzymatic peroxidation are summarized in Table 3. The highest activity was remarked for LPME (IC50 threescore.25 ± iv.7 μg/ml) (P < 0.01) of LP and is more stiff in inhibition of lipids peroxidation than other fractions. Antioxidant against lipids peroxidation was obtained for ascorbic acid (IC50 52.7 ± three.ii μg/ml).

Nitric oxide scavenging

Sodium nitroprusside in aqueous solution at physiological pH spontaneously produces nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Grries's reagent. Scavengers of nitric oxide compete with oxygen, leading to reduced product of nitrite ions. Overall, the LPME (IC50 55.4 ± 2.42 μg/ml) showed the highest nitric oxide scavenging (P < 0.01) ability compared to other fractions (Table 3) and ascorbic acid. When the scavenging abilaty was expressed as Trolox equivalent, information technology showed the methanol extracts was more potent than the other fractions.

Correlation with IC50 values of antioxidant and phytochemical constituents

Correlation analysis for phytochemical contents with IC50 values of radical scavenging and/or antioxidant ability of extract of LP and its various soluble fractions. The contents of phenolics and flavonoids showed meaning correlation (R2 0.6721–0.998) with DPPH, superoxide, hydrogen peroxide, phosphomolybdenum and ABTS radical scavenging (Table 4) while nonsignificantly correlated with scavenging of hydroxyl and nitric oxide radicals. In addition, IC50 of chelating power of atomic number 26 presented a significant correlation with flavonoids while non significant with phenolics.

Table 4 Correlations between the IC ₅₀ values of antioxidant activities, phenolics and flavonoids content of Launaea procumben s

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Word

Polyphenolic flavonoids are occurring ubiquitously in food and medicinal plants. They occur as glycosides and contain several phenolics hydroxyl groups. Many flavonoids are found to be strong antioxidants finer scavenging the reactive oxygen species because of their phenolics hydroxyl groups [thirteen]. Our study revealed the presence of six bioactive polyphenolic flavonoids (kaempferol, orientin, rutin, hyperuside, myricetin and quercetin) in LPME, which might play an important role in improving of oxidative stress [14]. Other studies reported the presence of the bioactive elective during chemical characterization of medicinal plants [15–eighteen]. The data of the present written report reveal the LPME contained notable amounts of phenolics compounds endowed with high antioxidant. These findings provide a proficient pharmacological logic for this found in renal injuries, hormonal and sexual disorders, and antimicrobial too as its use in folk and herbal medicine in Islamic republic of pakistan. It has been reported in many investigations that bioactive fractions of dissimilar medicinal plants having free radical scavenging and antioxidant, are used in many diseases like cancer, tissue inflammatory and cardiovascular disease [19–22]. Too, the number of publications on the health benefits of polyphenol has been increased [23, 24]. Diverse gratis radical scavenging methods used in this written report are elementary and have provided reproducible results showing antioxidant properties of diverse fractions of LP. The antioxidant capacity of different fractions observed in this experiment could be, considering of the presence of high phenolics compounds. LPME is more strong compared to other fractions and found in accordance with previous reports [25, 26], which accept shown that loftier total polyphenol content increases the antioxidant activity and proves a linear correlation between phenolics content and antioxidant activity. LPME exhibited a pregnant correlation every bit was reported by Bortolomeazzi et al.[27]. Phenolic compounds such as flavonoids, phenolics acid and tannins possess various biological activities such as anti-inflammatory, anticarcinogenic and antiatherosclerotic. The presence of these bioactive compounds might contribute to various scavenging effects of LP [28]. Free radicals of 1, 1-diphenyl 1-2-picrylhydrazyl (DPPH) are widely used for screening of medicinal plants to investigate their antioxidant potential. In these process-free, DPPH radicals when dissolves in methanol, give violet color in methanol solution. The results existed clearly betoken that in screening of various fractions of LP, methanol fraction had marked scavenging touch on with IC50 2.6 ± 0.004 at 50–250 μg/ml. Our results are supported by other investigation [29]. The potential of diverse fractions to scavenge free radical was as well assessed by their ability to quench ABTS, and depicts that LPME possessed IC50 (66.1 ± i.02 μg/ml) value, showing the strongest action fifty-fifty more than than reference compounds. Co-ordinate to Oszmianski et al.[30], the antioxidant activities against ABTS or DPPH were correlated to the concentration, chemical structures, and polymerization degrees of antioxidants. Hagerman et al.[31] take reported that the high molecular weight phenolics (tannins) have more abilities to quench costless radicals (ABTS) and their effectiveness depends on the molecular weight, the number of aromatic rings and nature of hydroxyl group'due south substitution than the specific functional groups. Free radical (ABTS·+) scavenging of LP fractions might be due to the presence of high molecular weight phenolics such as catechin, and rutin derivatives in improver to other flavonoids. The phosphomolybdate method has been routinely used to evaluate the full antioxidant capacity of extracts [32]. The results showed the methanol extracts of LP (IC50 64.27 ± 2.i μg/ml) indicated significant antioxidant activity, which was increased in a concentration-dependent manner. The results suggested that the strong antioxidant activity of extracts might be due to the presence of phenolics compounds nowadays in the extract [33]. Contempo investigation has shown that many flavonoids and related polyphenol contribute significantly to the antioxidant activity of many fruits such as ruddy grape, vegetables and medicinal plants [34]. Methanol extracts of LP also markedly scavenge hydroxyl, hydrogen peroxide, superoxide radicals and nitric oxide as well every bit possesses a strong metallic reducing power, in addition to bleach β-carotene, the meaning activity of LPME could exist due to the presence of bioactive flavonoids. Our results agree with the results of Shukla et al.[35] during the screening of in vitro antioxidant activity and total phenolics content of ethanol leafage extract of Stevia rebaudiana Bert. Similar investigation was reported in other studies [36]. Oxidative stress was characterized by increased lipids peroxidation and contradistinct nonenzymatic and enzymatic antioxidant. Cumulative show suggested that diverse enzymatic and nonenzymatic systems had been developed past mammalian cells to survive with ROS and other complimentary radicals. Methanolic extracts of LP markedly reduced lipid peroxidation comparatively to other fractions and reference compounds. Other studies accept like contribution during characterization of lipids peroxidation. Previous studies take shown that Mentha extracts be able to prevent the propagation of the lipids peroxidation process in a complex lipids matrix, such equally a foodstuff or biological membrane [37–39]. Flavonoids are a large group of compounds occurring ubiquitously in food plants. They occur every bit glycosides and comprise several phenolics hydroxyl groups in their ring structure, capable of antioxidant activities [xiii]. In our study, flavonoids showed a concentration dependent antioxidant activeness of different fractions of LP. Phenols are secondary metabolites in plants and are known to possess a wide range of therapeutic uses, such as antioxidant, antimutagenic, anticarcinogenic, free radical-scavenging and too subtract cardiovascular complications [40]. The scavenging ability of the phenols is mainly, because of the presence of hydroxyl groups. From the results obtained, information technology is inferred that total phenol contents were present in the reasonable amount in LPME and its derived fractions. A previous study also supports our results [41].

Materials and methods

Chemicals

Nitroblue tetrazolium (NBT), β-nicotinamide adenine dinucleotide reduced (β-NADH), 2-deoxy- D-ribose, linoleic acrid, ammonium thiocyanate, β-carotene, iii-(2-pyridyl)-5, 6 bis (iv-phenylsulfonic acid)-1,2,4-triazine (ferrozine), Phenazine methosulphate (PMS), 2,2-diphenyl-one-picrylhydrazyl (DPPH), ethylenediamine tetra acetic acrid (EDTA), rutin, ascorbic acid, gallic acid, potassium ferricyanide; trichloroacetic acrid (TCA), thiobarbituric acids (TBA) were obtained from Sigma Aldrich Chemical Co. (USA). All other reagents were of analytical grade.

Plant collection

Plants of LP at maturity were collected from Wah Cantt, city Rawalpindi (Islamic republic of pakistan). Plants were identified and a specimen was submitted at Herbarium of Islamic republic of pakistan, Quaid-I-Azam University Islamabad, Pakistan. Whole plant (leaves, stalk, flowers and seeds) were shades dried at room temperature for two weeks, chopped, ground mechanically of mesh size 1 mm.

Preparation of plant extracts

Five kg powder of Launaea procumbens was extracted twice in x liter of methanol with random shaking, after a week the excerpt was filtered through whatmann filter paper No. 45, filtrate was mixed and evaporated through rotary vacuum evaporator at 40°C to become 362 chiliad methanolic rough extracts (LPME). The crude excerpt was suspended in water and fractionated by liquid: liquid partition with solvents of increasing polarity; starting from n-hexane (23 m; LPHE), ethyl acetate (43 1000; LPEE) and chloroform (67 g; LPCE). All the fractions were stored at four °C for farther phytochemical and in vitro investigations.

Phytochemicals characterization

Determination of the full phenolics contents

Total phenolics contents (TPC) were estimated using the method of Singleton and Rossi [42]. Two hundred micro liters (i–5 mg/ml; dissolved in respective solvent) of each fraction was added in ten milliliter of one:10 folin-ciocalteu reagent and incubated for v min before the improver of seven ml of 0.115 mg/ml Na2CO3. The resulting solution was incubated a further 2 h before absorbance readings were taken at 765 nm. Gallic acid was used in the calibration curve. Results were expressed as mg gallic acid (GAE)/g dried institute extract. Data for each fraction was recorded in triplicate.

Determination of the total flavonoids

Full flavonoids content was adamant past using a method described by Sakanaka et al.,[43]. Briefly, 0.25 ml of each fraction (1–5 mg/ml; dissolved in respective solvent) and rutin standard solution (15–250 μg/ml) was mixed with one.25 ml of distilled water in a exam tube, followed by addition of 75 μl of a 5% (westward/v) sodium nitrite solution. After vi min, 150 μl of ten% (w/v) aluminum chloride solution was added, and the mixture was allowed to stand for a further 5 min before 0.v ml of 1 M NaOH was added. The mixture was made upwards to 2.v ml with distilled water and mixed well. The absorbance was measured immediately at 510 nm. The results of samples were expressed as mg of rutin equivalents of total dried fractions. All fractions were run in triplicate.

Loftier performance liquid chromatography'

1 gram powder was extracted with vi ml of 25% hydrochloric acid and xx ml methanol for 1 h.

The obtained extract was filtered to a volumetric flask. The residue was heated twice with 20 ml of methanol for 20 min. The combined excerpt was diluted with methanol to 100 ml. 5 ml portion of the solution was filtered and transferred to a volumetric flask and diluted with 10 ml of methanol. The sample (x μl) was injected into the HPLC apparatus. Samples were analyzed on Agilent HPLC. Separation was carried out through column (five μm; iv.6 × 150 mm, Agilent) with UV–vis detector. Solvent A (0.05% trifluoroacetic acid) and solvent B (0.038%trifluoroacetic acid in 83% acetonitrile (v/v) with the following slope: 0–5 min, fifteen% B in A, 5–10 min, seventy% B in A, 10–15 min, lxx% B in A are used for separation. The catamenia charge per unit was 1 ml/min and injection volume was 10 μl. Six different standards compounds (myricetin, catechin, vitexin, orientin, hyperuside, and rutin) were run for comparable detection and optimized. The scale curves were defined for each compound in the range of sample quantity 0.02–0.v μg. All samples were assayed in triplicate. All quantitative information were explained by annotator software.

Antioxidant assays

DPPH radical scavenging

The costless-radical scavenging activity was measured by using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. DPPH assay was performed according to the procedure as reported by Gyamfi et al.[44]. DPPH solution was prepared past dissolving 3.2 mg in 100 ml of 82% methanol. 2.8 ml of DPPH solution was added to glass vial followed past the addition of 0.2 ml of test sample solution, in methanol, leading to the final concentration of ane μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml and 100 μg/ml. Mixture of DPPH, and each fraction was shaken well and kept in the nighttime at controlled room temperature (25–28°C) for 1 h. Later on incubation change in color was measured at 517 nm. Mixture of 2.8 ml of 82% methanol and 0.2 ml of methanol were used as bare while 0.2 ml of methanol and 2.eight ml of DPPH solution were taken every bit control. The exam of each fraction was performed in triplicate. Percentage inhibition was measured according to following formula and IC50 value was calculated by graph pad prism software.

$$\%\text{\hspace{0.17em}scavenging}=\text{Abs}.\text{\hspace{0.17em}of\hspace{0.17em}command}–\text{Abs}.\text{\hspace{0.17em}of\hspace{0.17em}fraction}\times 100/\text{\hspace{0.17em}Abs}.\text{\hspace{0.17em}of\hspace{0.17em}control}$$

(i)

ABTS radical cation assay

ABTS radical cation assay was carried out using the protocol of Re et al.[45]. Co-ordinate to this protocol, ABTS (2, 20-azinobis-(3-ethylbenzothiazoneline-6-sulphonic acid, 7.4 mM) used every bit the gratuitous-radical provider, was treated with potassium persulfate (ii.45 mM) to produce free radicals. The solution was diluted to obtain an absorbance of ane.5–2.5 at 414 nm with 98% of ethanol, before used. Reagent (3 ml) was transferred to the glass cuvettes with one of them containing 3 ml ethanol every bit blank. The initial absorbance of the reagents in the glass cuvettes was recorded at 414 nm. 100 μl of each fraction (0.05–0.250 mg/ml) were transferred into the cuvettes containing the reagent, and the mixtures were shaken thoroughly. The mixture in the cuvette was examined afterwards ninety min using a UV–vis spectrophotometer. Antioxidant chapters of the ascorbic acid was also determined. The capability to scavenging the ABTS radical cation was calculated using the following equation;

$$\%\text{\hspace{0.17em}ABTS\hspace{0.17em}radical\hspace{0.17em}cation\hspace{0.17em}scavenging\hspace{0.17em}ability}=\left(\text{A}1-\text{A}2/\text{A}\mathrm{ane}\right)\times 100$$

(2)

Where A1 is the absorbance of the control (ABTS solution without test sample), and A2 is the absorbance in the presence of the test sample. The results reported are expressed equally their IC50 through Graph prism pad software.

Phosphomolybdenum assay

The antioxidant activity of fractions was evaluated by phosphomolybdenum method according to the procedure of Prieto et al.[32]. An aliquot of 0.1 ml of each fraction (dissolved in respective solvent) was combined in a vial with 1 ml of reagent solution (0.6 M sulphuric acrid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vial was capped and incubated in a h2o bath at 95°C for 90 min. After the incubation, samples were cooled to room temperature, and the absorbance of the mixture was measured at 765 nm against a bare. Percent inhibition was calculated past the following formula while IC50 was calculated through Graph prism pad software.

$$\%\text{inhibition}=\left(1–\text{absorbance\hspace{0.17em}of\hspace{0.17em}sample}/\text{absorbance\hspace{0.17em}of\hspace{0.17em}control}\right)\times 100$$

(3)

Antioxidant activity by β-carotene bleaching method

The antioxidant activity of each fraction was evaluated using the ß-carotene-linoleate model organisation, every bit described by Dominicus and Ho [46]. 2 mg of β-carotene were dissolved in 10 ml chloroform and 1 ml β-carotene solution was mixed with 20 mg of purified linoleic acrid and 200 mg of Tween twoscore emulsifiers. Chloroform was then evaporated under a gentle stream of nitrogen, and the resulting mixture was immediately diluted with l ml of distilled water. To an aliquot of v ml of this emulsion, 0.2 ml of each excerpt (0.05–0.250 mg/ml) or ascorbic acids were added and mixed well. The absorbance at 470 nm, which was regarded as t0, was measured, immediately, confronting a blank consisting of the emulsion without β-carotene. The capped tubes were placed in a water bath at fifty°C, and the absorbance was measured after every 15 min up to 120 min. For the positive control, sample was replaced with ascorbic acrid. A negative command consisted of 0.2 ml of distilled h2o or solvent instead of excerpt or reference antioxidant was used. All samples were assayed in triplicate. The antioxidant action (AA) was measured in terms of successful bleaching of β-carotene by using the following equation; $\text{AA}=(\left(\mathrm{i}–\left(\text{A}0–\text{At}/\text{A}0–\text{A}0\text{t}\right)\right)\times 100$

Where A0 and A0 are the absorbance values measured at zero times during the incubation for each fraction and control, respectively. At an A0t was the absorbance values measured for each fraction and control, respectively, after incubation for 120 min. The results were expressed equally IC50.

Superoxide radical scavenging activity

Superoxide radical scavenging activeness of each fraction was determined by the nitroblue tetrazolium reduction method [47]. I milliliter of nitroblue tetrazolium (NBT) solution (l Yard NBT in 100 mM phosphate buffer, pH vii.iv), 1 ml NADH solution (50 Chiliad NADH in 100 mM phosphate buffer, pH vii.four) and 0.one ml of the extracts (0.50–0.250 mg/ml) and ascorbic acrid (0.050–0.250 mg/ml) were mixed. The reaction was started by adding 100 μl of PMS solution (60 μM PMS in 100 mM phosphate buffer, pH seven.four) to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against bare samples, containing all the reagents except the PMS. The positive and negative controls were subjected to the aforementioned procedures as the sample, except that for the negative control, only the solvent was added, and for the positive command, sample was replaced with ascorbic acid. All measurements were made in triplicate. The abilities to scavenge the superoxide radical were calculated using the following equation;

$$\begin{array}{l}\%\text{\hspace{0.17em}superoxide\hspace{0.17em}radical\hspace{0.17em}scavenging\hspace{0.17em}activity}=\\ \left(\mathrm{i}-\text{absorbance\hspace{0.17em}of\hspace{0.17em}sample\hspace{0.17em}at}560\text{nm}/\text{absorbance\hspace{0.17em}of\hspace{0.17em}control\hspace{0.17em}at}560\text{nm}\right)\times 100\end{array}$$

(v)

IC50 was calculated through software.

Hydroxyl radical scavenging activity

The effect of extracts on hydroxyl radicals was assayed past using the deoxyribose method [48]. Solution of each fraction and ascorbic acrid (ASA) was prepared in methanol. The reaction mixture contained; 450 μl of 0.2 K sodium phosphate buffer (pH 7.0), 150 μl of 10 mM 2- deoxyribose, 150 μl of 10 mM FeSO_four-EDTA, 150 μl of x mM H₂O₂, 525 μl of H_twoO, and 75 μl of sample solution (0.050–0.250 mg/ml). The reaction was started past the add-on of H_iiO_ii. After incubation at 37°C for iv h, the reaction was stopped by adding 750 μl of 2.8% trichloroacetic acid and 750 μl of one% TBA in 50 mM NaOH, the solution was boiled for 10 min, and so cooled in h2o. The absorbance of the solution was measured at 520 nm. Ascorbic acid (0.05–0.250 mg/ml) was used as positive controls. The ability to scavenge the hydroxyl radical was calculated using the following equation;

$$\%\text{\hspace{0.17em}superoxide\hspace{0.17em}radical\hspace{0.17em}scavenging\hspace{0.17em}activity}=\left(\mathrm{one}-\text{absorbance\hspace{0.17em}of\hspace{0.17em}sample}/\text{absorbance\hspace{0.17em}of\hspace{0.17em}control}\right)\times 100$$

(6)

Hydrogen peroxide-scavenging activity

The ability of the extracts to scavenge hydrogen peroxide was determined according to the method of Ruch et al.[49]. A solution of hydrogen peroxide (2 mM) was prepared in 50 mM phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically at 230 nm absorption, using the molar extinction coefficient for H_iiO₂ of 81 mol^-i cm^-1. Samples of various fractions (0.050–0.250 mg/ml) and ascorbic acid (0.05–0.250 mg/ml) were transferred into the test tubes, and their volumes were fabricated up to 0.4 ml with 50 mM phosphate buffer (pH seven.4). Afterward addition of 0.6 ml hydrogen peroxide solution, tubes were vortex and absorbance of the hydrogen peroxide at 230 nm was adamant after 10 min, against a blank. 50 mM phosphate buffer without hydrogen peroxide was used every bit blank. Hydrogen per oxide scavenging power was calculated past following equation:

$$\text{Hydrogen\hspace{0.17em}peroxide\hspace{0.17em}scavenging\hspace{0.17em}activity}=\left(1–\text{absorbance\hspace{0.17em}of\hspace{0.17em}sample}/\text{absorbance\hspace{0.17em}of\hspace{0.17em}control}\right)\times 100$$

(vii)

IC50 was calculated through graph prism pad software.

Chelating activity on Fe2+

The extracts were assessed for their ability to compete with ferrozine for atomic number 26 (Two) ions in free solution. The chelating ability of ferrous ions by various fractions was estimated by the method of Dinis et al.[fifty]. Extracts (0.05–250 mg/ml), 2.five ml were added to a solution of 2 mM FeCl₂.4H₂O (0.05 ml). The reaction was initiated by the addition of v mM ferrozine (0.ii ml); the mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the solution was and then measured at 562 nm against the blank performed in the aforementioned way using FeCl₂ and water. EDTA (0.625–5 μg/ml) served as the positive control, and a sample without extract or EDTA served equally the negative control. All tests were run in triplicate and averaged. The per centum of inhibition of ferrozine-Fe² + complex formation was calculated using the formula:

$$\text{Chelating\hspace{0.17em}activity}\%=\left(1–\text{absorbance\hspace{0.17em}of\hspace{0.17em}sample}/\text{absorbance\hspace{0.17em}of\hspace{0.17em}control}\right)\times 100$$

(8)

Lipid peroxidation analysis

Lipid peroxidation assay was performed according to modified protocol of Banerjee et al.[51] to measure the lipid peroxide formed, using egg yolk homogenates as lipid-rich media [52]. Egg homogenate (0.5 ml of x%, v/5) and 0.ane ml of each fraction and ascorbic acid (0.v–0.250 mg/ml) was dissolved in respective solvent; were added to a test tube and made upward to 1 ml with distilled water. 0.05 ml of FeSO_four (0.07 M) was added to induce lipid peroxidation and incubated for 30 min. Then 1.v ml of 3.5 M acetic acid (pH adjusted to three.v with NaOH) and ane.5 ml of 0.06 M TBA in 0.04 One thousand sodium dodecyl sulphate and 0.05 ml of 1.2 Thou of TCA was added, and the resulting mixture was vortex and then heated at 95°C for lx min. To eliminate this not-MDA interference, another set up of samples was treated in the same style, incubating without TBA, to decrease the absorbance for fraction and reference compounds. After cooling, 5 ml of butan-1-ol was added to each tube and centrifuged at 3000 × g for 10 min. The absorbance of the organic upper layer was measured at 532 nm. Inhibition of lipid peroxidation (%) past the sample was calculated according to the following formula:

$$\%\text{inhibition}=\left(1-\text{East}/\text{C}\right)\times 100$$

(9)

Where C is the absorbance value of the fully oxidized command, and E is {(A532 + TBA)–(A532– TBA)}.

Nitric oxide scavenging activity

The nitric oxide scavenging activity was conducted based upon the method by Rai et al.[53]. 0.5 ml of 10 mM sodium nitroprusside in phosphate buffered-saline was mixed with 0.5 ml of different concentrations of the various fractions and control and incubated in the nighttime at room temperature for 150 min. After the incubation period, 1 ml of sulfanilic acid reagent (0.33% sulfanilic acid in 20% glacial acetic acid) was added to 0.5 ml of the reaction mixture. Afterwards 5 min incubation, i ml of 0.1% naphthyl ethylene diamine dihydrochloride was added and incubated for xxx min at 25°C. The absorbance of the chromophore formed was read at 540 nm. Ascorbic acid was used as positive command and results were expressed as percentage inhibition of nitric oxide. The nitric oxide scavenging activity of the extracts was also measured using the Trolox standard curve and results were expressed as mM Trolox equivalent antioxidant chapters (TEAC) per g dried fraction. All determinations were performed in triplicates.

Statistical analysis

All assays were carried out in triplicates, and results are expressed as hateful ± SD. ANOVA test was used to analyze the differences among IC50 of various fractions for different antioxidant assays, with least significance departure (LSD) P < 0.01 equally a level of significance. Experimental results were further analyzed for Pearson's correlation coefficient of phenolics, flavonoids with unlike antioxidant assays and tested for significance past educatee'south test (P < 0.05; P < 0.01). The IC50 values were calculated using graph pad prism software.

Conclusion

The results obtained in this written report take considerable value with respect to the antioxidant activities of LPME. The presence of these activities is attributed to the phenolics and poly phenolics compounds such as myricetin, catechin, vitexin, orientin, hyperoside, and rutin, revealed in HPLC. Our results suggested that the extract can exist utilized every bit an constructive and safe antioxidant source, every bit ethnomedicine and on a commercial basis for the development of drugs.

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Affiliations

1. Section of Biotechnology, Faculty of Biological Sciences, University of Science and Technology, Bannu, KPK 28100, Islamic republic of pakistan

   Rahmat Ali Khan & Mushtaq Ahmed

2. Department of Biochemistry, Faculty of Biological Sciences, Quaid-I-Azam Academy Islamabad, Islamabad, Pakistan

   Rahmat Ali Khan & Muhammad Rashid Khan

3. Botanical Sciences Division, Pakistan Museum of Natural History, Garden Avenue, Shakarparian, Islamabad, Pakistan

   Sumaira Sahreen

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Correspondence to Rahmat Ali Khan.

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RAK made a pregnant contribution to conquering of data, analysis, drafting of the manuscript. MRK and SS has fabricated a substantial contribution to formulation and design, interpretation of information, drafting and revising the manuscript for intellectual content. All authors read and canonical the final manuscript.

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Khan, R.A., Khan, M.R., Sahreen, Southward. et al. Assessment of flavonoids contents and in vitro antioxidant activity of Launaea procumbens. Chemistry Central Journal 6, 43 (2012). https://doi.org/10.1186/1752-153X-half-dozen-43

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• Received: 15 February 2012

• Accustomed: 04 May 2012

• Published: 22 May 2012

• DOI : https://doi.org/10.1186/1752-153X-half dozen-43

Keywords

• Launaea procumbens
• Scavenging of DPPH-free radicals
• Superoxide radicals
• HPLC
• Flavonoids

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