Antioxidant Activities of Root, Stem and Leaves of Vernonia cinerea (L) Less

INTRODUCTION

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.

RESULT

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.

Table 1

ANOVA for antioxidant activity by DPPH, ABTS and Phosphomolybdenum assays using different extracts from root, stem and leaf

Source	Df	MS	P
DPPH
Extracts	6	1026.522	< 0.05
Organs	2	938.114	< 0.05
Extracts x Organs	12	105.721	< 0.05
Error	42	3.539
ABTS
Extracts	6	930.941	< 0.05
Organs	2	488.777	< 0.05
Extracts x Organs	12	245.922	< 0.05
Error	42	19.859
Phosphomolybdenum
Extracts	6	.012	< 0.05
Organs	2	.022	< 0.05
Extracts x Organs	12	.008	< 0.05
Error	42	.000

Figure 1

Comparison of DPPH and ABTS radical scavenging activity (%) of different leaf extracts (A), stem extract (B) and root extract (C). Values are mean of three replicates ± Std. dev. Aq = Aqueous, Eth = Ethanol, Meth = Methanol, Ace = Acetone, EA = Ethyl acetate, PE = Petroleum ether, Chl = Chloroform. The error bars represent standard deviation. The activity is significant among the extracts and organs (p< 0.05)

Figure 2

Phosphomolybdenum assay using extracts from different plant parts. Values are mean of three replicates ± Std. dev. VL = Leaf Extract, VS = Stem Extract, VR = Root Extract, Aq = Aqueous, Eth = Ethanol, Meth = Methanol, Ace = Acetone, EA = Ethyl acetate, PE = Petroleum ether, Chl = Chloroform. The reduction of phosphomolybdenum complex is significant among organs and extracts (p < 0.05)

Figure 3

Correlation between % radical scavenging activity by DPPH and ABTS assays.

DISCUSSION

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.

The importance of use of combination of methods for determination of antioxidant activity has been suggested in several studies^19,20 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 investigators^9,10 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.

CONCLUSION

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.