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Comprehensive assessment of phenolics and antiradical potential of Rumex hastatus D. Don. roots
© Sahreen et al.; licensee BioMed Central Ltd. 2014
Received: 5 March 2013
Accepted: 5 February 2014
Published: 8 February 2014
Roots of Rumex hastatus (Polygonaceae) are traditionally used for the treatment of various ailments including liver and lung diseases. In this study, various solvent extracts of R. hastatus roots, like methanolic, n-hexane, ethyl acetate, chloroform, butanol and aqueous fractions were assessed through their antioxidant properties in vitro and determination of phenolic contents.
Several parameters like DPPH˙, ABTS˙+, ˙OH, H2O2, superoxide free radical scavenging, iron chelating power, reducing power, β-carotene bleaching power, antioxidant capacity and total phenolics and flavonoids were evaluated. High Performance liquid Chromatography (HPLC) was also considered.
Though all the fractions exhibited dose dependant activity. The samples with the highest activity were the butanol and methanol fractions in all the assays except hydrogen peroxide radical scavenging assay where chloroform fraction showed the highest scavenging aptitude. On the other hand, aquous fraction showed significant beta carotene linoleic acid, while n-hexane and ethyl acetate fractions exhibited a lesser antioxidant activity in all the assays. HPLC revealed the presence of rutin, luteolin-7-glucoside, vitexin and luteolin.
These results have to some extent substantiated the use of R. hastatus roots against different diseases, as an excellent basis of potential antioxidant due to the presence of sufficient amount of phenolics such as rutin and luteolin.
Rumex hastatus is suffrutescent richly branching shrub. It grows up to 90–120 cm tall and leaves with petioles of the same length as the blade; blade hastate, panicles terminal with erect-divergent, mostly simple branches, nut up to 2 mm long, brown, and long spindle-shaped roots. The plant is distributed in northern Pakistan, north east Afghanistan and south west of China, growing between 700-2500 m, sometimes grows as pure population . Researches have reported that root and the whole plant of R. hastatus is used as medicine. It is laxative, alterative, tonic, used in rheumatism , skin diseases, bilious complaints, piles, bleeding of lungs etc. . Plant is used as flavouring agent, carminative purgative and diuretic . Literature demonstrates that Rumex hastatus is traditionally used in the treatment of sexually transmitted diseases including AIDS . Oxidation is a source of energy for various bio processes but production of excessive oxygen free radicals causes oxidative damages that in turn initiate lipid peroxidation of protein and DNA [6, 7]. All the livings are gifted with enzymatic and non enzymatic antioxidant contents which protect the body from oxidative free radicals and balance them . In case of excessive free radicals large number of medicinal plants and fruits are used to minimize the effect due to the presence of bioactive antioxidant metabolites [9, 10]. Recently large number of research groups has focus on medicinal plant and their bioactive ingredients to replace costly synthetic drugs . Phenolic and polyphenolic constituents present in medicinal herbs leads retardation of lipid peroxidation and scavenging of free radicals [12, 13]. Our previous reports reveals that medicinal plant and their bioactive compounds play important role in free radicals scavenging and is used as therapeutic agent in the treatment of various disorders [14–16]. In our previous studies, leaves of R. hastatus were evaluated for phenolic compounds , so it is hypothesisized that roots being an ethnopharmacological part of R. hastatus, might have potent antioxidant properties against free radicals but no reports are available on the antioxidant activity of R. hastatus roots. Therefore, present study was conducted to explore the total phenolic content, HPLC characterizations and antioxidant activities of methanolic extracts and its various fractions through various in vitro models.
Plant collection and preparation of extract
During September 2010 R. hastatus was collected from Quaid-i-Azam University Islamabad, and Abbottabad of Northern Pakistan, respectively after identification by world renowed taxonomist Professor Dr. Mir Ajab Khan. Voucher specimens with Accession No. 27813 (R. hastatus) were deposited at the Herbarium of Pakistan Museum of Natural History, Islamabad for future correspondence. After complete cleaning of R. hastatus roots were cut into pieces and dried under shade. The dried samples were processed as mentioned [17, 18]. The dry extract obtained with each solvent was weighed and stored at 4°C for further investigations.
Determination of total phenolics
The total phenolics were assayed by the spectrophotometric method  using Folin- Ciocalteu’s phenol reagent at 750 nm. Gallic acid standard solution (0–100 mg/l) used as standard as shown as milligrams of gallic acid equivalents (GAE) per gram of dried sample.
Determination of total flavonoids
Total flavonoid content was determined following Yong et al. at 506 nm. Rutin was used as a standard and expressed as milligrams of rutin equivalents per gram of dried sample.
HPLC quantification of phenolic compounds
50 mg of each fraction of both plants were extracted with 6 ml of 25% hydrochloric acid and 20 ml methanol for 1 hr. The obtained extract was filtered to a volumetric flask. The residue was heated twice with 20 ml of methanol for 20 min to obtain the extract. The combined extract was diluted with methanol to 100 ml. 5 ml portion of the solution was filtered and then was transferred to a volumetric flask and diluted with 10 ml of methanol. The sample (10 μL) was injected into the HPLC apparatus.
The HPLC separation was performed using an Agilent HPLC system through column 20RBAX ECLIPSE, XDB-C18, (5 μm; 4.6 × 150 mm, Agilent USA) with UV–VIS Spectra-Focus detector, injector-auto sampler. The isocratic mobile phase, consisting of tetrahydro-furan/acetonitrile/ 0.05% phosphoric acid solution (20:3:77, v/v/v), was delivered at a flow-rate of1.0 mL/min with some amendments in time and wavelentgth . Prior to use the mobile phase was filtered through 0.45 mm Millipore membrane filters and degassed by sonication in an ultrasonic bath. Detection wavelength was set at 280 nm and the column temperature was maintained at 25ºC with injection volume of 10 μL.
Standard solution and calibration curves
Methanol stock solution containing rutin, luteolin, vitexin and luteolin-7-glucoside was prepared and diluted to appropriate concentrations for the construction of calibration curves. At least six concentrations of the solution were analyzed in triplicate, and then the calibration curves were constructed by plotting the peak area versus the concentration of each analyte detected by HPLC.
The LOD and LOQ
The stock solution containing rutin, luteolin, vitexin and luteolin-7-glucoside was diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions was injected into HPLC for analysis. The LOD and LOQ under the present chromatographic conditions were determined at S/N of 3 and 10, respectively.
Precision, repeatability, and accuracy
Intra- and inter-day variations were chosen to determine the precision of the developed assay. The known concentrations of rutin, luteolin, and luteolin-7-glucoside were tested. For intra-day variability test, the solution was examined in duplicates for three consecutive days while for inter-day variability test, the mixed standards solution was analyzed for six times within one day, Variations were expressed as the RSD.
To confirm the repeatability, BRR and MRR samples were extracted, respectively, and analyzed by HPLC as mentioned above. The RSD was used as the measurement of repeatability.
Identification and quantification
Where RT1 and RT2 and W1and W2 are the times and widths, respectively, of the two immediately adjacent peaks.
Each sample was dissolved in 95% methanol at a concentration1 mg/ml and then diluted to prepare the series concentrations for various antioxidant assays including DPPH , superoxide anion radical , total antioxidant efficacy , hydroxyl radical , hydrogen peroxide , ABTS radical cation  β-carotene bleaching , iron chelation  and reducing power scavenging activity was determined according to the method of Oyaizu .
All assays were carried out in triplicates and results are expressed as mean ± SD. Statistical comparisons were done with the ANOVA test. Differences were considered to be significant at p < 0.05. The EC50 values were calculated using the Graph Pad Prism 5 software.
Extraction yield, total phenolics and flavonoid contents of R. hastatus roots
Total phenolics, flavonoid and extraction yield of methanol extract and soluble fractions of R. hastatus roots
Total phenolics (mg GAE/g dry fraction)
Flavonoids (mg Rutin equivalent (mg RTE/g dry fraction)
Extraction yield (%)
111 ± 3.7c
38.6 ± 2.2c
12.5 ± 1.45d
8.9 ± 1.4a
4.2 ± 0.78a
4.5 ± 0.5b
11.3 ± 1.6a
7.0 ± 0.52a
5.7 ± 0.66b
81.9 ± 2.1b
37.7 ± 2.7c
3.2 ± 0.22a
128 ± 3.6d
42.6 ± 1.9d
11.3 ± 1.11d
88.1 ± 2.5b
22.4 ± 1.8b
9.1 ± 0.99c
HPLC quantification of flavonoids
Linearity and detection limits
Retention time, linear range, calibration curve, Correlation coefficient, LOD and LOQ of four flavonoids determined by HPLC (UV)
Linear range (μg/ml)
Corelation coefficient (r)
y = 8.623 × 105x + 946.965
y = 1.433x106x + 1064.141
y = 3.357x105x + 1085.241
y = 1.429x106x + 3818.831
Precision and recovery
Precision, Recovery and Content of four flavonoids in RH roots determined by HPLC (UV)
1.2 ± 0.12
0.78 ± 0.02
3.19 ± 0.24
0.87 ± 0.13
5.62 ± 0.47
6.12 ± 1.11
The results suggest that the activity of BRR and MRR is attributed to phenolic compounds and in particular to rutin, luteolin, luteolin-7–O-glucoside and vitexin however, unknown (peaks) compounds may also involve in the antioxidant activities of RH roots.
In vitro antioxidant assays
DPPH radical scavenging activity
Antioxidant effect (EC 50 ) of methanol extract and its various fractions of R. hastatus roots
Scavenging ability on DPPH radicals
Scavenging ability on super oxide radicals
Total antioxidant capacity
Scavenging ability on hydroxyl radicals
Scavenging ability on H2O2radicals
Scavenging ability on ABTS radicals
β-carotene bleaching inhibition
84.16 ± 3.2c
21.21 ± 0.5a
61.87 ± 2.4c
90.21 ± 2.1c
128.01 ± 2.1c
126.67 ± 2.9d
333.11 ± 2.3b
> > 250d
> > 250e
> > 8000f
242.78 ± 5.4c
99.23 ± 2.3d
62.28 ± 3.3e
45.85 ± 1.2b
85.29 ± 2.1d
75.11 ± 1.7b
136.09 ± 4.1c
138.09 ± 3.3e
1233.24 ± 4.5e
68.04 ± 1.7b
43.19 ± 2.4b
21.78 ± 0.8a
36.56 ± 1.6b
77.02 ± 1.5b
96.17 ± 1.12b
84.14 ± 2.6b
600.75 ± 4.1c
85.28 ± 1.9c
56.49 ± 0.9d
23.48 ± 0.4a
63.92 ± 1.9c
99.11 ± 3.3c
143.06 ± 1.8d
114.89 ± 3.6c
800.28 ± 2.2d
19.59 ± 0.8a
22.36 ± 0.6a
20.79 ± 0.4a
29.78 ± 1.1a
23.04 ± 0.7a
65.23 ± 1.5a
72.41 ± 2.5a
233.72 ± 1.9a
19.31 ± 0.7a
27.10 ± 0.5a
Superoxide radical scavenging activity
The superoxide radical scavenging activity of different fractions of R. hastatus was compared with ascorbic acid ranging from 25–250 μg/ml. EC50 values in hydroxyl scavenging activities were in the order of BRR > MRR > AFC > CRR > ERR > HRR (Table 4). All of the fractions had a scavenging activity on the superoxide radicals in a dose dependent manner.
Phosphomolybdate assay (Total antioxidant capacity)
Total antioxidant capacity depicts that different fractions of methanol extract of roots of R. hastatus can be ranked in the order of BRR > MRR > ARR > CRR > ELR > HRR. The EC50 values for the BRR and MRR was the same i.e. 21.78 ± 0.8 μg/ml, 21.21 ± 0.5 μg/ml, respectively while for the HRR it was >250 μg/ml (Table 4). The results obtained imply that the BRR and MRR have a remarkable ability to act as antioxidant.
Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity can be ranked as BRR > MRR > ARR > CRR > ERR > HRR. Scavenging activity of all the extracts was found to be low when compared to ascorbic acid. The EC50 values of scavenging hydroxyl radicals for the BRR was 36.56 ± 1.6 μg/ml, while for the HRR was >250 μg/ml (Table 4).
Hydrogen peroxide radical scavenging activity
Extracts from R. hastatus roots were capable of scavenging hydrogen peroxide in a concentration-dependent manner (25–250 μg/ml). The CRR and BRR fractions were equally potent in scavenging hydrogen peroxide, by 61.6 ± 1.21% and 57.1 ± 3.17% at a concentration of 100 μg/ml, respectively, while ERR and HRR were considerably less effective hydrogen peroxide scavengers (24.11 ± 1.22% and 27.03 ± 1.07%) at the same concentration. As compared with the EC50 values, the hydrogen peroxide- scavenging activities of CRR (75.11 ± 1.7 μg/ml) and BRR (77.02 ± 1.5 μg/ml) were comparable, and more effective (p < 0.05) than that of ERR (> > 250 μg/ml) and HRR (>250 μg/ml) (Table 4). The scavenging abilities on hydrogen peroxide were in descending order of CRR > BRR > MRR > ARR > ERR > HRR.
ABTS radical scavenging activity
ABTS radical scavenging ability of samples can be ranked as BRR > MRR > CRR > ARR > ERR > HRR. The percentage inhibition was 87.01 ± 1.12% , 92.18 ± 0.08% and 82.03 ± 2.24% for the BRR, MRR and CRR, respectively while for ARR, ERR and HRR inhibition was 78.01 ± 3.19%, 47.43 ± 1.45% and 41.1 ± 2.6% at a concentration of 500 μg/ml. BRR exhibited the highest radical scavenging activities comparative to HRR and ERR. The EC50 values obtained for the BRR (96.17 ± 1.12 μg/ml) was significantly different (p < 0.05) from the EC50 values obtained for the ERR (>250 μg/ml) and HRR (> > 250 μg/ml), which were comparable (Table 4) with reference chemicals.
β-carotene bleaching assay
With regard to the β-carotene bleaching assay, the antioxidant activity of samples can be ranked as BRR > ARR > MRR > CRR > ERR > HRR. At 0.1 mg/ml, β-carotene bleaching inhibitions were 54.11 ± 2.33% , 48.09 ± 1.44%, 40.78 ± 3.42%, 39.1 ± 2.56%, 18.15 ± 2.13% and 15.37 ± 2.51% for BRR, ARR, MRR, CRR, ERR and HRR, respectively. At 0.5 mg/ml the inhibition was increased to 81.34 ± 2.34%, 73.12 ± 1.19%, 78.62 ± 2.67%, 72.18 ± 1.36%, 34.49 ± 2.34% and 29.09 ± 1.45% for the above order. The EC50 values of BRR and ARR were 84.14 ± 2.6 μg/ml and 114.89 ± 3.6 μg/ml, respectively (Table 4) which was comparable with catechin.
Iron chelating activity
Reducing power activity
Correlation of EC50 values of antioxidant activities and phytochemical contents
Correlation 1 between EC 50 values of antioxidant activities and total phenolics and flavonoids of R. hastatus root extract and its various soluble fractions
EC50 of DPPH radical scavenging ability
EC50 of superoxide radical scavenging ability
EC50 of antioxidant capacity
EC50 of hydroxyl radical scavenging ability
EC50 of hydrogen peroxide radical scavenging ability
EC50 of ABTS radical scavenging ability
EC50 of β-carotene bleaching inhibition
EC50 of chelating power
As the plant derived polyphenols exhibit typical inhibitory trend against in vitro and in vivo oxidative reactions  due to redox properties therefore, it can be stated that tested plant samples may have important role to scavenge free radicals as they contain substantial quantity of phenolics and flavonoids. According to Sharififar et al. dietary intake of flavonoid-containing foods was suggested to be of benefit as free radical stabilizers can replace the synthetic antioxidants by retarding lipid peroxidation. However, results obtained in the present study revealed that phenolics and flavonoids are main constituents of the plant having pharmacological tendency. Flavonoids are of considerable interest because of their possible inverse association with various chronic diseases like coronary heart disease  and several forms of cancer especially breast cancer . In HPLC profile of R. hastatus roots, luteolin and rutin were recorded in higher amounts hence, could be consumed as a new source of bioactive compound. Qur studies revealed that type and amount of flavonoid compounds can be important evidence in the identification and evaluation of the best fraction. Data shows that HPLC profile of R. hastatus roots provide support to in vitro assays in which above mentioned fractions appeared to maintain strong antioxidant effects.
Antioxidant activity in an in vitro experiment is considered as the first step to point out potential health power of these fractions. Indeed, the study verified the role of phenolic and flavonoid contents extracted through different solvents against oxidative injuries. Thus, our results suggested that the extract can be exploited as an efficient and valuable antioxidant source, as some of the fractions showed highest scavenging ability than that of synthetic compounds. DPPH, a stable free radical, changes its color from violet to yellow after reduction by antioxidant or radical scavenger donating hydrogen- or electron . The DPPH free radical has been commonly used to assess the antioxidative potential of plant extracts. It has been recommended that extracts rich in phenolics and flavonoids are involved in several biological activities including antioxidant ones. The study presented that power of fractions from roots of R. hastatus to scavenge DPPH radicals was associated with phytochemicals extracted by different solvents indicating that BRR and MRR can also possibly act as primary antioxidant. The superoxide anion is the more frequently produced free radical. Under oxidative stress, intense increase in superoxides results in cell and DNA damage which ultimately causes several pathological diseases . It was therefore anticipated to calculate the relative ability of fractions to quench the superoxide free radicals. Several in vitro methods are accessible to generate superoxide free radicals . In the present study, BRR and MRR behaved as strong superoxide anion quenchers among all the tested samples. The quenching ability of fractions may be the result of reactive concentration of bioactive compounds like phenolics and flavonoids and may help to put off oxidative damage of the major bio-molecules. Total antioxidant capacity of fractions/extracts has been estimated by phosphomolybdate method . In this method, antioxidants reduce the Mo (VI) into Mo (V). The results obtained imply that BRR and MRR have notable antioxidant ability as compared to reference (ascorbic acid) antioxidant. This strong activity of various fractions of roots of R. hastatus might be a certificate for antioxidant behaviour of phenolic compounds. Reports of Sharififar et al. also revealed that total antioxidant activity of medicinal plants is interlinked with flavonoids. Hence it offers an opportunity to develop less contemptible natural antioxidants. The hydroxyl radical is being concerned as highly reactive and detrimental species for about every molecule of biological system cause pathophysiological diseases. It has a potential to react with lipids, proteins and nucleotides of DNA causing oxidative damage. It was therefore proposed by Babu et al.  that hydroxyl radical scavenging ability is frankly allied with antioxidant activity. In the present study, BRR and MRR also reacted as strong hydroxyl quenchers. Thus, hydroxyl radical scavenging ability seems to be directly linked with prevention of lipid peroxidation and reduction of chain reactions.
Hydrogen peroxide itself is not very reactive, but it may produce hydroxyl free radicals that are very toxic to cells . Inspite of this, previous researchers have focused on the hydrogen peroxide scavenging ability to find out the antioxidant status of the plant extract or pure compounds. Results showed the most valuable hydrogen peroxide scavenging ability of BRR and CRR as their values were analogous to that of reference compound. According to Hagerman et al. phenolics with high molecular weight have additional capacity to quench free radicals like ABTS and their efficiency is more allied with number of aromatic rings and nature of hydroxyl group’s substitution as compare to functional groups. It can therefore be estimated that free radical (ABTS) scavenging action of BRR, MRR and CRR might be accredited with high molecular weight phenolics in addition to the flavonoids. β-carotene bleaching inhibition was accounted in different solvent extracts of dill (Anethum graveolens) flower . The efficacy of R. hastatus root to hamper oxidation of linoleic acid emulsion is an indication of complex composition of fractions to interact with emulsion components. This data suggested that BRR and ARR have a notable propensity to scavenge free radicals that result in more stable non-reactive substances and to terminate radical chain reactions. Iron chelating data shows that various fraction possibly have talent to act against oxidative damage by chelating iron ions that may otherwise participate in decomposition of metal- catalyzed hydro peroxide and Fenton-Type reactions . The iron sequestering frequency of various fractions measured at different concentrations proves that BRR and MRR may act as chelating agents against metallic ions. Accordingly it can be entailed that endogenous chelating agents like phenolics and flavonoids may be credited for iron chelating properties of various fractions. In addition, some phenolic compounds having well oriented functional groups possess the ability to protect against oxidative damage by chelating metal ions.
In the reducing power assay, donation of an electron is required to convert Fe+3/ferric cyanide complex to ferrous form that necessitate antioxidant. The total ferrous complex can be scrutinized by computing the development of Perl’s Prussian blue at 700 nm. Data suggested that BRR and MRR have a noteworthy power to react with free radicals by converting them into more stable substances and to stop radical chain reactions. Activity may be endorsed with the collective antioxidant effects of phytochemicals especially phenolics and flavonoids. The results indicate that phenolics and flavonoids are responsible for the antioxidant activities of fractions of R. hastatus roots, and decorated the importance of phenolic compounds in the antioxidant measures of plant fractions. A noticeable correlation among different tests confirmed the viability and consistency of selected antioxidant assay systems. Our results are in agreement with Ao et al. who reported strong relationship with DPPH and ABTS as compare to β-carotene. Correlation studies of R. hastatus roots proved the role of phytochemicals especially phenolics and flavonoids in antioxidant potential of plant. The results suggest that the activity of BRR and MRR is attributed to phenolic compounds and in particular to rutin, luteolin, luteolin-7–O-glucoside and vitexin however, unknown (peaks) compounds may also involve in the antioxidant activities of RH roots.
In conclusion, the proposed HPLC method showed a good linearity, precision, repeatability, accuracy and recovery for the determination of four active compounds (rutin, luteolin, vitexin and luteolin-7-glucoside) and could be used for the quantitative analysis of RH roots. In this study, determination of phenolics and flavonoids, HPLC quantification and 9 antioxidant activity method with 6 extraction systems of different polarities, i.e., n-hexane, ethyl acetate, chloroform, butanol, methanol and water were compared. To our best knowledge this is the first record on the antioxidant potential of R. hastatus roots.
We have demonstrated that R. hastatus roots extracts were capable of inhibiting and directly quenching free radicals to terminate the radical chain reaction which might be attributed to the presence of flavonoid contents such as luteolin, rutin, vitexin and luteolin-7-glucoside.
We are very thankful to Higher Education Commission (HEC) Pakistan for provision research funds.
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