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.
KeywordsCarissa opaca leaves Free radical scavenging Solvent–solvent extraction Total phenolics and flavonoids
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.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Rehman EU (2006) Indigenous knowledge on medicinal plants, village Barali Kass and its allied areas, District Kotli Azad Jammu and Kashmir. Pak. Ethnobot Leafl 10:254–264Google Scholar
- Khan RA, Khan MR, Sahreen S (2011) Protective effect of Sonchus asper extracts against experimentally—induced lung injuries in rats: a novel study. Exp Toxicol Pathol. (in press) Google Scholar
- Jeruto P, Lukhoba C, Ouma G, Otieno D, Mutai C (2008) An ethnobotanical study of medicinal plants used by the Nandi people in Kenya. J Ethnopharm 116:370–376View ArticleGoogle Scholar
- Khan RA, Khan MR, Sahreen S, Bukhari J (2010) Prevention of CCl4-induced nephrotoxicity with Sonchus asper in rat. Food Chem Toxicol 23:1304–1321Google Scholar
- Kareru PG, Kenji GM, Gachanja AN, Keriko JM, Mungai G (2007) Traditional medicine among the Embu and Mbeere peoples of Kenya. Afric J Trad Compl Alter Med 4:75–86Google Scholar
- Khan MR, Haroon J, Khan RA, Bokhari J, Rashid U (2011) Prevention of KBr O3-induced cardiotoxicity by Sonchus asper in rat. J Med Plants Res 12:2514–2520Google Scholar
- Afolayan AJ, Jimoh FO (2008) Nutritional quality of some wild leafy vegetables in South Africa. Intl J Food Sci Nutr 60:424–431View ArticleGoogle Scholar
- Middleton E, Kandaswami C, Theoharides TC (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol Rev 52:673–751Google Scholar
- Alpinar K, Ozyurek M, Kolak U, Guclu K, Aras C, Altun M, Celik SE, Berker KI, Bektasoglu B, Apak R (2009) Antioxidant capacities of some food plants wildly grown in Ayvalik of Turkey. Food Sci Tech Res 15:59–64View ArticleGoogle Scholar
- Amic D, Davidovic-Amic D, Beslo D, Trinajstic N (2003) Structure–radical scavenging activity relationship of flavonoids. Croat Chem Acta 76:55–61Google Scholar
- Balasundram N, Sundram K, Samman S (2006) Phenolic compounds in plants and agro industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem 99:191–203View ArticleGoogle Scholar
- El-Zalabani SM, Mahmoud II, Ahmed FI, Shehab NG (1999) Protein carbohydrate, mineral and vitamin contents of Sonchus oleraceus L. growing in Egypt. J Pharm Sci 23:46–54Google Scholar
- Sahreen S, Khan MR, Khan RA, Shah NA (2013) Effect of C. opaca leaves extract on lipid peroxidation, antioxidant activity and reproductive hormones in male rats. Lipids Health Dis. 20(12):90. doi:10.1186/1476-511X-12-90 View ArticleGoogle Scholar
- Sakanaka S, Tachibana Y, Okada Y (2005) Preparation and antioxidant properties of extracts of Japanese persimmon leaf tea (kakinoha-cha). Food Chem 89:569–575View ArticleGoogle Scholar
- Hagerman AE, Riedl KM, Jones GA, Sovik KN, Ritchard NT, Hartzfeld PW (1998) High molecular weight plant polyphenolics (tannins) as biological antioxidants. J Agri Food chem 46:1887–1892View ArticleGoogle Scholar
- Liu X, Zhao M, Wanga J, Yangb B, Jiang Y (2008) Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China. J Food Comp Anal 21:219–228View ArticleGoogle Scholar
- Ozsoy N, Can A, Yanardag R, Akev N (2008) Antioxidant activity of Smilax excelsa L. leaf extracts. Food Chem 110:571–583View ArticleGoogle Scholar
- Singleton VL, Rossi JA (1996) Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Viticult 16:144–153Google Scholar
- Gyamfi MA, Yonamine M, Aniya Y (1999) Free radical scavenging activity of medicinal herb of Ghana: Thonningia sanguinea on experimentally induced liver injuries. Gen Pharmacol 32:661–667View ArticleGoogle Scholar
- Re R, Pellegrini N, Proteggente A, Pannala A, Yong M, Rice-Evas C (1999) Antioxidant activity applying an improved ABTS radical cation decoloursation assay. Free Rad Biol Med 26:1231–1237View ArticleGoogle Scholar
- Nishikimi M, Rao NA, Yagi K (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 46:849–854View ArticleGoogle Scholar
- Ruch RJ, Cheng SJ, Klaunig JE (1989) Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechin isolated from Chinese green tea. Carcinogenesis 10:1003–1008View ArticleGoogle Scholar
- Nagai T, Myoda T, Nagashima T (2005) Antioxidative activities of water extract and ethanol extract from field horsetail (tsukushi) Equisetum arvense L. Food Chem 91:389–394View ArticleGoogle Scholar
- Dinis TCP, Madeira VMC, Almeida LM (1994) Action of phenolic derivatives (acetaminophen, salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch Biochem Biophys 315:161–169View ArticleGoogle Scholar