Evaluation of antioxidant profile of various solvent extracts of Carissa opaca leaves: an edible plant
© The Author(s) 2017
Received: 10 April 2017
Accepted: 14 July 2017
Published: 18 August 2017
Carissa opaca leaves were conventionally recommended by local hakims in Pakistan for curing various human diseases including renal, hepatic and jaundice. In this work we arranged to study the antioxidant status of various fractions of C. opaca leaves through nine multifaceted assay systems.
Various fractions were prepared through solvent–solvent extraction technique on the basis of their polarity. The fractions were screened via different free radicals viz; DPPH·, ABTS·+,OH·, O2·, iron chelating and hydrogen peroxide assays. Total concentrations of phenolic content (TPC) and flavonoids were studied.
Various fractions of C. opaca leaves showed significant activities against the tested reactive free radicals. The C. opaca was shown to have the highest TPCs with lowest EC50 values for the DPPH·, ABTS·+ radical scavenging capacities and iron chelating scavenging efficiency, moreover, C. opaca had best activities in scavenging of superoxide radicals and hydrogen peroxide as well as potently scavenged the hydroxyl radicals.
These results suggest the potential of C. opaca leaves as a medicine against free-radical-associated oxidative damage.
Plants are well-known excellent perspectives for the discovery of new therapeutical products. In recent years, an ample interest has been developed in finding natural antioxidants from commonly available wild plants, fruits and vegetables that were generally mistreated [1–3]. It is believed that they possess a remarkable potential to overwhelm the deadly diseases of modern world. Numerous reports of crude extracts and pure natural compounds have been appeared for antioxidant and radical-scavenging activities [4–7]. Phenolic compounds which are secondary metabolites in plants are one of the most widely occurring groups of phytochemicals that exhibit antiallergenic, antimicrobial, antiartherogenic, antithrombotic, anti-inflammatory, vasodilatory and cardio protective effects [8, 9]. Due to the presence of the conjugated ring structures and hydroxyl groups; many phenolic compounds have the potential to function as antioxidants by scavenging or stabilizing free radicals involved in oxidative processes through hydrogenation or complexing with oxidizing species that are much stronger than those of vitamins C and E [10, 11].
Carissa opaca Stapf ex Hanes, is a 2–3 m tall evergreen shrub containing glabrous or puberulous branches with opposite and ovate glabrous leaves, hard and sharp spines arising between the petiole. Flower color white with 12 mm long slender corolla tube. Edible berry fruits with dark purple color after ripening. Distribution of plant in Pakistan is from Punjab to Himalayas up to 6000 ft, in Murree. The leaves are used traditionally for the treatment of asthma, cardiac dysfunction, hepatitis and jaundice. Due the lack of scientific studies of its potential pharmacological properties, the objective of this study was to evaluate the antioxidant activity through direct free radical scavenging methods and also elucidate total phenolic content (TPC) and polyphenolic flavonoids constituents of various fractions of C. opaca leaves.
Total phenolics, total flavonoids and % yield contents (TPC)
Antioxidant effect (EC50) on DPPH radicals, superoxide radicals, total antioxidant capacity and hydroxyl radicals of methanol extract and soluble fractions of C. opaca leaves
Scavenging ability on DPPH radicals
Scavenging ability on superoxide radicals
Total antioxidant capacity
Scavenging ability on hydroxyl radicals
58 ± 1.6c
93 ± 1.92b
30 ± 1.5b
22 ± 1.4b
358 ± 4.92e
135 ± 3.6c
22 ± 1.3b
444 ± 4.11f
206 ± 4.23e
18 ± 0.7a
132 ± 3.6c
18 ± 1.1a
170 ± 2.7d
229 ± 5.4f
156 ± 3.9d
18 ± 0.9a
38 ± 1.33b
159 ± 2.45d
81 ± 2.7c
18 ± 0.89a
16 ± 1.6a
21.86 ± 1.3a
22 ± 1.8a
30 ± 1.1c
18 ± 1.19a
Invitro antioxidant activities
Antioxidant effect (EC50) on hydrogen peroxide radicals, ABTS radicals, inhibition of β carotene and chelating power of methanol extract and soluble fractions of C. opaca leaves
Scavenging ability on hydrogen peroxide radicals
Scavenging ability on ABTS radicals
β-carotene bleaching inhibition
155 ± 3.2b
104 ± 4.6b
49 ± 1.9b
19 ± 1.1a
133 ± 3.5c
157 ± 3.12b
16 ± 0.98a
225 ± 6.39c
176 ± 4.0d
145 ± 4.3b
73 ± 2.9c
160 ± 4.7b
181 ± 3.1d
16 ± 1.1a
70 ± 3.2a
50 ± 2.3b
243 ± 2.5c
187 ± 3.8d
137 ± 3.76d
23.04 ± 1.7a
67 ± 2.5a
38 ± 2.8a
20 ± 1.2a
29.04 ± 1.5a
Correlation of EC50 values of antioxidant activities and phytochemical contents
Correlations between the EC50 values of antioxidant activities and phenolic and flavonoids content of C. opaca leaves
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
Carissa opaca leaves is used ethno pharmacologically for the treatment of various complaints. The therapeutic benefit of medicinal plants is usually contributed to their antioxidant properties. The biochemical investigation reported that C. opaca leaves constitute of antioxidant compounds such as carotenoids, catechin, rutin, quercetin and other phenolics [12, 13]. Moreover, C. opaca leaves activities against oxidative stress, antibacterial and antitumor were yet to be explored. Different free-radical generating systems were used to assess the free-radical scavenging and reducing properties of the crude polar and non-polar extracts of C. opaca leaves along with evaluation of the total phenolic content. Quantitative estimation proved that the C. opaca leaves possesses the highest concentration of phenolic compounds in methanol fraction of the extract. Similar results were described by other studies in the literature for other extracts of plants . The C. opaca leaves provided us with plentiful of different sorts of polyphenolic compounds as an incredible source of antioxidant, exhibited by the remarkable EC50 values in different extracts. The observed differential scavenging activities of the extracts against various systems may be referred to the different mechanisms of the radical antioxidant reactions in the different assays. Hagerman et al.  have reported that the high molecular weight phenolics (tannins) have more abilities to quench free radicals (ABTS·+) and their effectiveness depends on the molecular weight, the number of aromatic rings and nature of hydroxyl group’s substitution than the specific functional groups. Free radical (ABTS·+) scavenging activity of C. opaca leaves extracts might be due to the presence of high molecular weight phenolics such as catechin, and rutin derivatives. The C. opaca leaves extracts exhibit remarkable H2O2 and OH· radical scavenging capacity rendering, their utilization in different ailments associated with oxidative stress [16, 17]. Recent investigations have shown that many flavonoids and related polyphenols contribute significantly to the antioxidant activity of medicinal plants. Our results revealed that there is a strong and significant correlation between TPC and DPPH· free radical scavenging activity and H2O2 scavenging activity for the C. opaca leaves extracts, while the other assays have non-significant correlation with the TPC. This could be due to the difference in the stoichiometry of reactions between the antioxidant compounds in the extracts and the various radicals, which may be inferred as a reason for the difference in their scavenging potential. The diversity in radical scavenging shown in these assays may also be due to factors like stereo selectivity of the radicals or the differential solubility that may be justified in case of crude extracts, which contain a variety of antioxidants.
Materials and methods
Plant collection and extraction
Total phenolic and flavonoids contents
The total phenolic content was determined using the method  with certain modifications. Calibration curve was prepared by mixing methanolic solution of gallic acid (1 ml; 0.025–0.400 mg/ml) with 5 ml Folin–Ciocalteu reagent (diluted tenfold) while total flavonoids content was determined by using a method described . All fractions were run in triplicate.
In vitro antioxidant activity
The free-radical scavenging activity of the various fractions, gallic acid and ascorbic acid was measured with the stable radical diphenylpicrylhydrazyl (DPPH) in terms of hydrogen-donating or radical-scavenging activity  with some modifications. ABTS assay was performed according to the protocol  while superoxide scavenging was determined by the nitroblue tetrazolium reduction method . The scavenging capacity for hydrogen peroxide was measured according to the method . The effect of extracts on hydroxyl radicals was assayed by using the deoxyribose method . The extracts were assessed for their ability to compete with ferrozine for iron (II) ions in free solution. The chelating ability of ferrous ions by various fractions was estimated by the method .
EC50 was carried out using graph prism pad software. Experimental results were further analyzed for Pearson correlation coefficient between TPCs, flavonoids and different antioxidant assays and tested for significance by Student’s t test (P < 0.05). SPSS ver. 14.0 (Chicago, IL, USA) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations.
This study revealed that the activities are may be due to the presence of bioactive phenolic and flavonoid contents.
RAK made a significant contribution to acquisition of data, analysis, drafting of the manuscript. MRK has made a substantial contribution to conception and design, interpretation of data, drafting and revising the manuscript for intellectual content. SS participated in the design and collection of data and analysis. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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