The metabolic process of human body in its natural course creates several reactive oxygen species (ROS) in the form of superoxide anion (O2-.), hydrogen peroxide (H2O2) and hydroxyl radical (OH·). The generation of excess ROS leads to development of many diseases which include insulin resistance, Diabetes mellitus, sepsis, Obesity1 and Alzheimer’s.2 In order to treat several such diseases an antioxidant based therapeutics is suggested. Such prominent remedies are commonly used for treatment of diabetic neuropathy,3 venous insufficiency4 and neuroprotection5
There is a rising interest among the researchers to search and extract antioxidants from various medicinal plants, vegetables and fruits. These antioxidants are free radical scavengers that stop free radical chain reactions and support such treatments. Ethnomedicinal plants are good sources of antioxidants and are a potential source in the development of drugs and the treatment of different ROS generated diseases. Many compounds such as Vitamin C, Vitamin E, carotenoids and polyphenols from different plant sources have capacity to scavenge free radicals and are well recognized as antioxidants.6
Vernonia cinerea (L) Less plant is ethnomedicinally important. It is traditionally used as febrifuge, diaphoretic, diuretic, antispasmodic and anthelmintic.7 Various parts of the plant are reported to contain sterols such as stigmasterols, sitosterols and spinasterols; triterpenoids like amyrin and lupeol; sesquiterpenes, glycosides and flavonoids.7-10 Experimental evidences strongly suggest the anti-inflammatory and cytotoxic properties of plant extracts.8,11 The earlier experiments have investigated the antioxidant activities of V. cinerea plant using whole plant and leaf extract from single solvent.11,12
In the present study we considered the importance of medicinal properties and bioactive compounds of V. cinerea and carried out a comprehensive evaluation of antioxidant activities of root, stem and leaf extracts.
Based on earlier studies, it was recommended to use a multiple methods to determine antioxidant activities13 so as to understand different mechanisms involved in antioxidation process. We used three different assays and compared the antioxidant activities of root, stem and leaf extracts prepared using both polar and non-polar solvents. The LC- MS profiling identified compounds from the extracts showing significant antioxidant activity. This identification of phytoconstituents will help in isolation of compounds which are natural sources of antioxidant molecules.
MATERIALS AND METHODS
2, 2 - Diphenyl- 1- Picrylhydrazyl (DPPH), 2, 2- Azinobis (3 ethylbenzothiazoline 6 sulfonic acid) (ABTS) were purchased from Sigma Aldrich USA. Other chemicals and solvents used were of analytical grade and purchased from local distributor.
Collection of Plant material
Plants of Vernonia cinerea were uprooted from different regions of Pune (18°32’17.6”N73°48’20.4”E and 18°31’35.0”N 73°50’43.5”E). Plant was authenticated from BSI and Voucher specimen was submitted with ARGVEC3 code. The parts of plant, root, stem and leaves were separated and dried in shade.
Preparation of plant extracts
Air dried plant parts were powdered and stored in air tight containers. The powder was extracted by cold maceration technique at room temperature for 24 hours with constant shaking. The extracts were prepared in range of polar and nonpolar solvents as water, ethanol, methanol, acetone, ethyl acetate, petroleum ether and chloroform for extraction of different types of phytoconstituents. The proportion of plant material to solvent was 1:10(w/v).The extracts were filtered, centrifuged and dried by evaporation. The dried extract were suspended in DMSO to obtain yield of 10mg/ml of extract and stored at −20°C until they were used. All 21 extracts were evaluated for their potential for antioxidant activity using three assays.
DPPH radical scavenging assay
The assay was carried out according to Blois (1958).14 DPPH (0.1 mM) was prepared in 100% methanol. To 1 ml of DPPH solution, 200 μl of extract was added in a range of concentration (50 μg to 250 μg/ml). The mixture was incubated for 30 minutes in dark. After dilution, the absorption of reduced DPPH was measured at 517 nm (UV-VIS spectrophotometry). Methanolic DPPH was used as control. The Radical scavenging activity (RSA) was calculated in percentage by following formula.
The results were also reported as EC50 which is amount of antioxidant required to decrease the DPPH· concentration by 50%.EC50 values were calculated from regression analysis and expressed as μg dry weight equivalents per ml sample. The experiments were performed in triplicates. The results are expressed in average ± standard deviation.
ABTS radical scavenging assay
The assay was carried out according to the procedure given in review article.15 Reagent was prepared by mixing 10 ml of ABTS (14 mM ) and 10 ml potassium persulphate (4.9 mM). The mixture was kept in the dark for 16 h. The absorbance of the reagent was adjusted with distilled water to 0.700 ± 0.02 at 734 nm and then used for the assay. A stable ABTS radical cation is produced by oxidation of ABTS by potassium persulphate. 200 μl of plant extract in a range of concentration (50 μg to 250 μg) was added to 1 ml reagent mixture. After incubation for 10 minutes the reduction of radical cation was measured at 734 nm.
The results were expressed in terms of RSA in percentage. The results are also reported as EC50 which is amount of antioxidant required to reduce the ABTS radicals by 50%. ABTS reagent without plant extract was used as control whereas Ascorbic acid as positive control.
Phosphomolybdenum reduction potential of plant extracts were determined according to Prieto et al.16 Reagent was prepared by mixing ammonium molybdate (4 mM), sulphuric acid (0.6 M) and monosodium phosphate (28 mM) and final volume was made as per requirement.
Assay mixture contained 200 μl of plant extract in a range of concentration (50 μg to 250 μg), and 1ml reagent solution. The mixture was incubated at 95°C for 90 minutes. The absorbance of green complex was measured at 695 nm. The reagent itself was used as blank and Ascorbic acid as positive control.
The result is represented in terms of the number of microgram equivalents of ascorbic acid per milligram of plant extract.
LC- MS profiling of crude extracts showing maximum antioxidant activity was carried out.LC- MS analysis was performed on Agilent 1260 binary LC system and separated on Agilent Zorbax Extend C 18 RRHT column (50x2.1 mm, 1.8 μm) using aqueous 0.1% formic acid (mobile phase A) and Acetonitrile (mobile phase B). A stepwise gradient of solvent A and solvent B was applied at flow rate of 0.3 ml/min for 30 minutes. The capillary voltage, cone voltage, fragmentor voltage were 4kV, 45V and 170 V, respectively. The gas temperature was set at 325°. Electroscopy mass spectra data were recorded on a positive ionization mode. The data was acquired at scan rate of 3Hz in mass range 100- 1000 m/z and analyzed with Mass hunter qualitative software and METLIN database.
DPPH radical scavenging assay
DPPH radical scavenging activity was measured spectrophotometrically after incubation of plant extracts with DPPH for 30 minutes in dark. In the chosen plant extracts the highest DPPH radical scavenging activity was observed in methanolic extract (45.12% EC 50 312.06 μg/ml) among various leaf extracts; ethanolic (54 % EC 50 164.53 μg/ml) and methanolic (52.67% EC 50 232.33 μg/ml) among various stem extracts; methanolic (37.04% EC 50 442.27 μg/ml) among root extracts. As compared to root extracts, the antioxidant activity of stem and leaf extracts were significantly different (p 0.05) (Figure 1).
ABTS radical scavenging activity
ABTS radical scavenging potential of leaf methanolic extract (45.56 % EC 50 355.06 μg/ml) was highest among other extracts. Whereas in stem, it was highest in methanolic extract (56.13% EC 50 264.12 μg/ml) compared to other extracts. Methanol extract (52.12 % EC 50 257.28 μg/ml) of root has shown maximum antioxidant activity. There was significant difference in antioxidant activity among extracts of different plant parts (p< 0.05) (Figure 1).
The reduction of Mo was highest with methanolic leaf extract (129.56 μmolAAE/mg of extract), methanolic stem extract (307.37 μmolAAE/mg of extract) and aqueous root extract (368.29 μmolAAE/mg of extract). Phosphomolybdenum reduction potential of different extracts from root, stem and leaf are shown in Figure 2.
LC- MS profiling
MS data analysis using Mass Hunter and METLIN database search reveals presence of various primary and secondary metabolites and intermediate compounds. The identified secondary metabolites predominantly belongs to sesquiterpenes, diterpenes, triterpenes, steroids, glucuronides, quinones and phenols. The comparison of the phytochemicals in three different extracts along with molecular formula, molecular mass and m/z is shown in Table 2.
The aim of the present study was to determine and compare the antioxidant activity of different organs of V. cinerea which is an ethnomedicinal plant and identification of probable bioactive phytoconstituents with antioxidant properties. We have carried out comprehensive study by choosing polar and nonpolar extracts of root, stem and leaves of a plant body.
The antioxidant assays carried out using DPPH and ABTS radicals. They are Single Electron Transfer (SET) reactions where reaction is either electron transfer or through hydrogen atom transfer.17 ABTS·+ method is considered to be more sensitive since they react rapidly with lipophilic and hydrophilic antioxidants. The reduction of DPPH is quicker or slower process as it depends on the conformation of antioxidant compounds.18 We observed that the reaction of antioxidants with DPPH· was slower than that with ABTS·+ suggesting use of ABTS radicals is more suitable for determination of radical scavenging potential of crude extracts which contain mixture of compounds.
There was difference in the antioxidant activity in DPPH and ABTS methods but similarity was observed in the type of extract showing maximum radical scavenging activity. There is no strong correlation (Figure 3) between % scavenging measured by both tests (Pearson’s correlation coefficient r=0.558) but is significant at p<0.01. These observations indicate that the behavior of antioxidant compounds towards DPPH and ABTS is different or the difference in the result is due to requirement of different incubation time by two different assays.
Results of phosphomolybdenum assay differ from DPPH and ABTS methods. The formation of green complex was less in extracts of nonpolar solvent compared to extracts of polar solvent. Aqueous and acetone extracts of root have exhibited significant antioxidant activity compared to other extracts. In phosphomolybdenum assay the antioxidant activity was higher in root extracts whereas in DPPH and ABTS assays radical scavenging activity was high in stem and leaf extracts. This result indicates that the reduction of Mo depends on the types and amount of antioxidants.
In three different assays there was significant difference in antioxidant activities by extracts (Table 1) prepared in polar and nonpolar solvents (p<0.05) indicating difference in the types and amounts of compounds extracted by different types of solvents.
|Molecules||Formula||Mass||m/z||VS Me*||VR Me*||VL Me*|
|Orsellinic acid, Ethyl ester||C10H12O4||196.07||179.06||+||-||+|
|DL-7-hydroxy stearic acid||C18H36O3||300.26||283.26||-||+||-|
|Duartin, Dimethyl ether||C20H24O6||360.156||361.163||-||-||+|
|Anandamide (20:2, n-6)||C22H41NO2||351.312||334.309||-||-||+|
|Sulindac sulfide glucuronide||C26H25FO8S||516.12||517.13||+||+||+|
|Oleananoic acid acetate||C32H52O4||500.38||483.38||-||+||-|
The importance of use of combination of methods for determination of antioxidant activity has been suggested in several studies19,20 and it is clear from the above discussion that antioxidant activity differs with the method used and also depends upon nature of antioxidants.
In view of potential antioxidant activity of methanolic extracts of root, stem and leaves; they were subjected to LC-MS screening for identification of phytoconstituents. MS analysis reveals the presence of different classes of secondary metabolites along with primary metabolites. The metabolic diversity was observed and most prominent compounds were types of isoprenoids, steroids and phenols. In our study stem and leaf methanol extracts exhibited more radical scavenging activity compared to root methanol extracts. It can be correlated with diversity of different classes of compounds identified in stem and leaf methanol extracts. Most of the antioxidant studies are focused on isolation, identification and quantification of phenolic compounds which have proved to be having strong antioxidant property.21,22 Different hydroxybenzoic acid and hydroxycinnamic acid derivatives are identified in methanol extracts of V. cinerea. It is most likely that these could be bioactive components. But the antioxidant potential of terpenoids,23,24 quinones,25 steroids,26 and Chromone derivatives27 has also been demonstrated in various studies and these classes of compounds are identified in the screened extracts. There are several reports where plants rich in terpenes and phenolics are used as food additives owing to their antioxidant and antimicrobial properties.28 In our study the compounds identified are predominantly terpenes along with other classes. Sesquiterpenes and triterpenoids have been reported and isolated from V. cinerea by different investigators9,10 and also been reported to have anti-inflammatory and cytotoxic properties.8,11 These findings suggest that terpenes, phenolic compounds could be alone or by synergistic process responsible for antioxidant activity.
The antioxidant activity of different extracts from root, stem and leaves was comparable and significant. Use of different methods in determination of antioxidant activity suggested that the behavior of phytoconstituents is different with different oxidants. The root, stem and leaves of V. cinerea are source of antioxidant compounds and can be used in herbal preparations required for the treatment of ROS mediated diseases.
The study provides comprehensive analysis of antioxidant activities of V. cinerea using different organs and extraction of different phytoconstituents. The untargeted metabolic analysis using LC-MS could reveal presence of different classes of therapeutically active secondary metabolites involved in antioxidation. Methanolic extracts of plant organs are potent in antioxidant activities. Determination of antioxidant activities using different methods is useful to understand the mechanism of antioxidant activities and nature of antioxidants.