Research Article

The Analysis of Antioxidant Activity and Phenolic Contents of Selected Medicinal Plants of Mizoram  

Lalrinzuali K. , Vabeiryureilai M. , Jagetia G.C.
Department of Zoology, Mizoram University, Aizawl, 796004, India
Author    Correspondence author
Genomics and Applied Biology, 2015, Vol. 6, No. 11   doi: 10.5376/gab.2015.06.0011
Received: 12 Nov., 2015    Accepted: 30 Jan., 2016    Published: 28 Mar., 2016
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Lalrinzuali K., Vabeiryureilai M., and Jagetia G.C., 2015,THE ANALYSIS OF ANTIOXIDANT ACTIVITY AND PHENOLIC CONTENTS OF SELECTED MEDICINAL PLANTS OF MIZORAM, India, Genomics and Applied Biology, 6(11): 1-12 (doi: 10.5376/gab.2015.06.0011)

Abstract

The plants have been an integral part of human civilization and plants have played an important role in human healthcare since the time immemorial. The induction of oxidative stress has been a major problem as excess oxidative stress has been linked to almost all disease in humans. The present study attempted to evaluate the antioxidant potential and total phenol contents of certain medicinal plants including Schima wallichi (DC) Korth., Milletia pachycarpa Benth. stem bark, the leaves of Eleagnus caudata Schlecht, Dysoxylum gobara Buch.-Ham and the fruit of Castanopsis indica (Roxb.) A.DC. in vitro. The analysis of DPPH free radical scavenging activity has shown that chloroform and ethanol extracts of all these plants were able to scavenge free radicals in a concentration dependent manner. The maximum scavenging activity was reported for Eleagnus caudata, Milletia pachycarpa and Schima wallichi followed by Castanopsis indica and Dysoxylum gobara, which had the least activity. The ethanol extract had higher antioxidant activity than the chloroform extract. This was reflected in the total phenol contents that increased in a concentration dependent manner for both the chloroform and ethanol extracts of all plants. However, the amount of total phenols was greater in the ethanol extracts than that of chloroform extracts which were also least in Dysoxylum gobara. The antioxidant activity was directly proportional to the total phenol contents of a plant species. Our study demonstrates that all the five plants showed DPPH scavenging activity and the Schima wallichi showed highest effects as indicated by presence of higher amount of total phenols, which may have contributed to this effect.

Keywords
DPPH; Total phenols; Schima wallichi; Eleagnus caudata; Castanopsis indica; Milletia pachycarpa; Dysoxylum gobara

1 Introduction
The evolution of aerobic respiration in life and use of oxygen has come with a cost to all living organisms that use oxygen to produce chemical energy for various processes. The consumption of oxygen during respiration generates free radicals and reactive oxygen species. The free radicals are principally produced by various biochemical processes that are an indispensible part of aerobic life and metabolism (Halliwell and Gutteridge, 1999). The amount of free radical production and other reactive species is tightly controlled in living organisms, by a complex web of antioxidant defense mechanisms that help to minimize oxidative damage to different important biomolecules. The utilization of molecular oxygen for respiration may be toxic under certain conditions despite the fact that it is very essential for aerobic life. This phenomenon has been termed as the oxygen paradox (Gilbert, 2000). In a healthy human body the generation of reactive oxygen species and their neutralization is properly maintained as the endogenous enzymes scavenge and minimize the formation of reactive oxygen species as and when they are formed however, this phenomenon is not 100% efficient (Halliwell, 2001), especially when a burst of free radicals are produced, which may overwhelm the endogenous antioxidant system.


ROS have been traditionally regarded as byproducts of aerobic metabolism, and mitochondrial respiration is considered to be the major site of intracellular source of accidental ROS production.  Principally mitochondria use oxygen and produce the superoxide anion (O2.–) and the presence of MnSOD catalyzes the dismutation of O2.– into H2O2, the more stable toxic product and it may be converted into the highly reactive  hydroxyl radicals (.OH) in the presence of metals by Fenton reaction (Murphy, 2009; Lemire et al., 2013). Normally 1% to 3% of the oxygen consumed by the human body is converted into reactive oxygen species including superoxide anion (Burton, 2009). When not neutralized, the ROS accumulate in the cells and cause various forms of reversible and irreversible oxidative modifications of proteins, lipids and DNA that eventually lead to loss of molecular functions. Cells can normally defend themselves against ROS damage through the use of specific ROS reducing mechanisms that can be enzymatic (dismutases, catalases and peroxidases) or non-enzymatic (vitamins A, C and E, urate and bilirubin) (Giorgio et al., 2007).  Dietary and endogenous antioxidants prevent cellular damage by reacting with and eliminating oxidizing free radicals (Lamson and Brignall, 1999). Despite the fact that body has inbuilt different endogenous enzymes, which are able to scavenge free radicals, it is possible that endogenous system is unable to cope with excess production of free radicals and may require supplementation from outside (Bouayed and Bohn, 2010; Otohinoyi et al., 2014).


Various pharmacophores including butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG) and tert-butyl hydroquinone (TBHQ) are the most common antioxidants used however, their safety is in doubt due to toxic side effects associated with them (Branien, 1975; Sun and Fukuhara, 1997) indicating the need for more safe and nontoxic substitutes. Fruits and vegetables have been used by humans since time immemorial and they are rich sources of various antioxidants as they contain phenolics and polyphenols (Pan et al., 2008; Shukla et al., 2009). Apart from antioxidant activity phenols and poly phenols are also antimutagenic and anticarinogenic (Jonfia-Essien et al., 2008). The phenolic compounds can act as antioxidants by donating hydrogen to highly reactive radicals, thereby preventing further radical formation (Lapornik et al., 2005). Plant based antioxidants are believed to be less toxic, safer and even more reliable than their synthetic counterparts used for healthcare (Benli et al., 2008). These facts inspired us to investigate antioxidant potential of different plants including Eleagnus caudata, Milletia pachycarpa, Schima wallichi Castanopsis indica, and Dysoxylum gobara, possessing various medicinal properties in vitro by assessing DPPH scavenging activity.


2 Materials and Methods
2.1 Chemicals

Gallic acid, trolox and 1,1-Diphenyl-2-picrythydrazyl (DPPH) were procured from Sigma-Aldrich Co. Bangalore, India), whereas Folin-Ciocalteu reagent, sodium carbonate, methanol and other routine chemicals were procured form Merck India Ltd., Mumbai, India.


2.2 Collection of plant materials
The non-infected stem bark of Schima wallichi (DC) Korth. (Family: Theaceae), Milletia pachycarpa Benth. (Family: Fabaceae), the leaves of Eleagnus caudata Schlecht. (Family: Elaeagnaceae), Dysoxylum gobara Buch.-Ham (Family: Meliaceae) and the fruit of Castanopsis indica (Roxb.) A.DC. (Family: Fagaceae) were collected during the dry season from different parts of Mizoram. The plant specimens were identified by the Department of Aromatic plants and Horticulture, Mizoram University, Aizawl. The herbarium specimen of each plant sample has been deposited at the Department of Zoology, Mizoram University. The stem bark, leaves or fruits were inspected visually for infection, washed thoroughly with clean water and allowed to shade dry at room temperature in the dark, clean and hygienic conditions. The dried plant material was powdered using an electrical grinder at room temperature.


The powdered material from each plant was sequentially extracted with petroleum ether, chloroform and ethanol according to increasing polarity using a Soxhlet apparatus at their respective boiling points until they became colorless. The liquid extracts, except the petroleum ether extracts were filtered and concentrated by evaporating their liquid contents using rotary vacuum evaporator. They were then concentrated and stored at -70°C until further use.


2.3 DPPH radical scavenging activity
The principle for DPPH free radical reduction is that the antioxidant reacts with the stable free radical DPPH and converts it into 2,2-diphenyl-1-picryl hydrazine by donating an electron or hydrogen. The discoloration is due to the increasing scavenging of DPPH radicals. The test was carried out according to Leong and Shui, 2002 after minor modification. Briefly, to the different concentrations (10, 50, 100, 200, 500, 1000, 2500 and 5000 μg/ml) of the chloroform and ethanol extracts of Schima wallichi, Milletia pachycarpa, the leaves of Eleagnus caudata, Dysoxylum gobara and the fruit of Castanopsis indica (0.5 ml each), 1 ml of methanol solution of 0.1 mM DPPH was added. After mixing thoroughly, the mixture was allowed to stand in the dark for 30 mins and the absorbance was measured at 523 nm using methanol for the baseline correction. The results were compared with that of the control prepared as above without sample. Radical scavenging activity has been expressed as a percentage and was calculated using the following formula:
% Scavenging= (Acontrol-Asample)/ AsampleX100
Where Asample is the absorbance of the test sample and Acontrol is the absorbance of the control.


2.4 Total phenolic content

The total phenolic contents were determined using Folin-Ciocalteau reagent (McDonald et al., 2001). Briefly, different concentrations (100, 200, 500, 1000 and 2500 mg) of various extracts (0.5 ml) or gallic acid (standard phenolic compound) was mixed with Folin-Ciocalteau reagent (5 ml, 1:10 diluted with distilled water) and aqueous 1 M Na2CO3 (4 ml). The mixture was allowed to stand for 15 minutes and the total phenolic contents were measured at 756 nm with a UV-VIS double beam spectrophotometer (Systronic, Ahmedabad, India). The total phenol contents are expressed in terms of gallic acid equivalent (mg/gm of extracts), which is a common reference compound.


3 Results
The results are expressed as percent scavenging or trolox equivalent scavenging and total phenolic contents as gallic acid equivalent in figure 1 to 6. Table 1 shows the medicinal uses of the plants analyzed.

 

 
Table 1 Traditional uses of selected medicinal plants

 
3.1 Medicinal applications
Elaeagnus caudata Schlecht. (Family: Elaegnaceae): The fruit is taken as a health tonic (Kala, 2012). The extract of the fruit or stem bark is mixed with Piper longum and is taken daily for 2-3 weeks to cure jaundice and other liver troubles (Shankar et al., 2012). The roots are boiled and the water is taken orally to expel the retained placenta. Crushed root juice is taken to ease labor and as a treatment after child birth. Leaf infusion is taken orally to strengthen uterus function after child birth (Rai and Lalramnghinglova, 2011). About 5 ml of the fresh root extract is diluted in about 100 ml of water and taken once a week during pregnancy to prevent miscarriage (Rout et al., 2012).


Milletia pachycarpa Benth. (Family: Fabaceae): It is a large deciduous climbing shrub (Sawmliana, 2013). Bark paste is applied on the skin infections and itches until the disease is cured. Bark as well as the roots and pods are also used as fish poison (Rout et al., 2012). The juice of the crushed root is also applied on the mange of pigs (Sawmliana, 2013). It is also used as a blood tonic and to induce the growth of red blood cells in Chinese traditional medicine (Haifan and Zhang, 1996).


Schima wallichii (DC) Korth. (Family: Theaceae): The powdered fruit is applied on scorpion sting, bites of centipedes and spiders. The juice of the bark is used for the treatment of chronic ulcer and fresh wounds (Sawmliana, 2013). Decoction or infusion of leaf is used as an antiseptic (Kumar, 2002; Lalramnghinglova, 2003). Fruit decoction is used for snakebite and insect bite (Lalfakzuala, et al., 2007). The bark powder is taken with water for the treatment of gastritis (Joshi et al., 2011). The bark of this plant is traditionally used as antipyretic, antiseptic, anthelmintic and wound healing agent (Dash and Ghosh, 2013).


Castanopsis indica (Roxb.) A.DC. (Family: Fagaceae): It is commonly known as Indian chestnut tree and it is found throughout the Himalayan region of North East India, Bangladesh, Bhutan, Laos, Myanmar, Nepal, Sikkim, Thailand and Vietnam (Malla and Chhetri, 2009). The seeds are consumed raw in Nepal (Shrestha and Dhillio, 2012) and Mizoram (Kar et al., 2013). Different parts of the plant are traditionally used for the treatment of stomach disorder, skin diseases, diarrhea, chest pain and headache (Manandhar, 2002). A decoction of the leaves is applied to treat stomach disorder and skin diseases (Malla and Chhetri, 2009) and the powdered leaves are used as remedy of indigestion. The resin is given to treat diarrhea and the leaf paste is applied for headache. The bark paste is also applied on the chest to control chest pain (Joshi et al., 2011).


Dysoxylum gobara Buch.-Ham (Family: Meliaceae): Decoction of leaf and buds is used to treat diarrhoea and dysentery (Mahanti, 1994; Lalramnghinglova, 2003; Lalfakzuala et al., 2007). In Manipur, the leaves are boiled and taken in excess to cure diarrhea (Rout et al., 2010). The tender leaves and flowers are cooked and eaten as the vegetable. The decoction of leaves is used as a remedy for food poisoning, diarrhea and dysentery (Sawmliana M., 2013).


3.2 DPPH scavenging
The chloroform extract of Eleagnus caudata showed a concentration dependent rise in the DPPH scavenging activity. The maximum DPPH inhibition for chloroform extract was at 200 μg/ml, which remained almost same at 500 μg/ml and declined thereafter (Figure 1).

 

 

Figure 1 Scavenging of DPPH free radicals by the chloroform extract of different plants

Note: <: Castanopsis indica<: Dysoxylum gobara; <: Eleagnus caudata; <: Milletia pachycarpa and <: Schima wallichi

 
The ethanol extract of Eleagnus caudata showed a similar pattern for DPPH scavenging however the maximum effect was observed for 500 μg/ml that remained almost similar up to a concentration of 5000 μg/ml (Figure 2).

 

 

Figure 2 Scavenging of DPPH free radicals by the ethanol extract of different plants

Note: <: Castanopsis indica; <: Dysoxylum gobara; <: Eleagnus caudata; <: Milletia pachycarpa and <: Schima wallichi

 
Assessment of chloroform extract of Milletia pachycarpa, Schima wallichi, and Castanopsis indica showed a concentration dependent rise in the DPPH scavenging and the maximum scavenging was observed at 2500 µg/ml, whereas this concentration was 5000 μg/ml for Dysoxylum gobara (Figure 1). The ethanol extracts of Milletia pachycarpa, Schima wallichi, Dysoxylum gobara and Castanopsis indica also showed a concentration dependent rise in the DPPH scavenging up to a certain concentration and declined thereafter (Figure 2). The highest scavenging was observed at 500 μg/ml for Milletia pachycarpa and Schima wallichi, whereas 2500μg/ml for Dysoxylum gobara (Figure 2).


The analysis of DPPH scavenging in respect of trolox equivalent was different as maximum scavenging activity was observed at concentration of 5000 μg/ml for chloroform extracts of all five plants (Figure 3).

 

 

Figure 3 Scavenging of DPPH free radicals in relation to trolox equivalent by the chloroform extract of different plants

Note: <: Castanopsis indica; <: Dysoxylum gobara; <: Eleagnus caudata; <: Milletia pachycarpa; <: Schima wallichi

 
The ethanol extracts of Milletia pachycarpa, Schima wallichi and Eleagnus caudata showed a concentration dependent rise in the trolox equivalent scavenging activity up to 500 μg/ml thereafter there was not any significant alteration in the DPPH scavenging activity up to 5000 μg/ml for these plants (Figure 4). However, a constant rise in DPPH trolox equivalent scavenging was observed for Castanopsis indica and Dysoxylum gobara (Figure 4).

 

 

Figure 4 Scavenging of DPPH free radicals in terms of trolox equivalent by the ethanol extract of different plants

Note: <: Castanopsis indica; <: Dysoxylum gobara; <: Eleagnus caudata; <: Milletia pachycarpa and <: Schima wallichi

 
The order of scavenging activity was observed as follows: MPE (91.31 ± 1.55% at 5000 µg/ml) > SWE (90.39 ± 2.05% at 500 µg/ml) > ECE (85.14 ± 1.54% at 1000 µg/ml) > MPC (81.41 ± 1.1% at 2500 µg/ml) > ECC (78.38 ± 1.36%  at 500 µg/ml) > CIE (77.02 ± 0.9% at 2500 µg/ml) > SWC (72.5 ± 1.9% at 2500 µg/ml) > CIC (70.56 ± 1.011% at 5000 µg/ml) > DGE (65.50 ± 2.35% at 2500 µg/ml) > DGC (62.56 ± 1.55% at 5000 µg/ml) (Figure 1).


3.3 Total phenols
The total phenol contents of chloroform extracts of Milletia pachycarpa, Schima wallichi, Eleagnus caudata, Castanopsis indica and Dysoxylum gobara showed a concentration dependent rise up to 2500 μg/ml (Figure 5). The highest total phenols were present in Milletia pachycarpa followed by Eleagnus caudata and Schima wallichi. The least total phenols were detected in Dysoxylum gobara (Figure 5).

 

 

Figure 5 Total phenolic contents in the chloroform extract of different plants

Note: <: Castanopsis indica; <: Dysoxylum gobara; <: Eleagnus caudata; <:Milletia pachycarpa and <: Schima wallichi

 
The trend of presence of total phenol contents in ethanol extracts of all five plants was almost similar to that of chloroform extract except that the amount detected was significantly higher than the chloroform extract (Figure 6). A concentration dependent increase of total phenol contents was observed for all the extracts. The ethanol extract of Schima wallichi contained the highest phenolic contents whereas the chloroform extract of Dysoxylum gobara was found to have the lowest content. The order of the phenolic content observed was as follows: SWE (500.01 ± 0.28) > MPE (426.52 ± 0.13) > CIE (129 ± 0.09) > ECE (120.91 ± 0.07) > MPC (86.45 ± 0.07) > ECC (83.43 ± 0.07) > DGE (77.61 ± 0.45) > SWC (72.36 ± 0.02) > CIC (51.50 ± 0.05) > DGC (48.87 ± 0.18) at the concentration 2500 µg/ml.

 

 

Figure 6 Total phenolic contents in the ethanol extract of different plants

Note: <: Castanopsis indica; <: Dysoxylum gobara; <: Eleagnus caudata; <:Milletia pachycarpa and <: Schima wallichi

 
4 Discussion
Plants synthesize several chemicals for different purposes in the form of secondary metabolites (Pichersky and Gang, 2000). These chemicals find different uses in plants. The presence of these biomolecules makes plants very useful in humans. They are useful as medicines, poisons, colours and other applications. Polyphenols present in plants play an important role in human health. Therefore, the present study was aimed to evaluate the total phenol contents of certain medicinal plants including Eleagnus caudata, Milletia pachycarpa, Schima wallichi, Castanopsis indica and Dysoxylum gobara and subsequently analyze their antioxidant potential in vitro by evaluating their DPPH scavenging activity.


Generation of free radicals is an important even in the oxidative metabolism. It is well known that superoxide anion radical and hydrogen peroxide are the byproducts of utilization of free oxygen in the mitochondria. If not neutralized these are converted into more toxic and highly reactive hydroxyl radicals in the presence of metals by Fenton reaction and this vicious cycle generates oxidative stress (Murphy, 2009; Lemire et al., 2013). Apart from this several chemical, physical and environmental agents also induce oxidative stress which constantly threaten the integrity of the human genome (Krystona et al., 2011). The oxidative stress has been linked to all most all diseases including cardiovascular and neurodegenerative disorders, arthritis, autoimmune diseases, diabetes, and cancer. The oxidative stress is also indicated in aging (Halliwell, 1994; 2012; Pham-Huy et al., 2008). Therefore, a need has been felt for the agents that could neutralize the endogenous as well as exogenous free radicals. Several synthetic antioxidants including butylated hydroxytoluene and butylated hydroxyanisole, tert-butylhydroquinone, propyl gallate, octyl gallate, nordihydroguaiaretic acid, 4-Hexylresorcinol, allopurinol, tocopherol, ascorbic acid and deferoxamine have been most widely used as food additives and treatment of diseases (Delanty and Dichter, 2000; Gilgun-Sherki et al., 2003; Carocho and Ferreira, 2013). However, these antioxidants are not without adverse side effects (Branien, 1975; Sun and Fukuhara, 1997).


The α, α-diphenyl-β-picrylhydrazyl (DPPH) is a stable free radical as it contains a spare electron and the molecule does not dimerize unlike other free radicals (Blois, 1958). The free electron of DPPH is delocalized and gives violet colour at 520 nm. The combination of DPPH with a molecule that can donate hydrogen is reduced and loses violet colour and becomes a stable species (Contreras-Guzman and Strong, 1982). This determines the ability of any substance as an antioxidant. Therefore, it is used as an indicator of antioxidant potential of any substance that is able to donate hydrogen to DPPH. This assay is frequently used to estimate the antioxidant activity of any substance.


The natural antioxidants may be useful as they may have fewer side effects or no side effects, because of their biologic origin. Eleagnus caudate scavenged the DPPH free radicals in a concentration dependent manner indicating its antioxidant potential. The other species of Eleagnus including Eleagnus umbellata have been reported to scavenge DPPH radical (Khattak, 2012). This activity may be due the presence of polyphenol in it. The antioxidant nature and presence of polyphenols correlates well with its medicinal applications.


The chloroform and ethanol extracts of Milletia pachycarpa did inhibit the generation of DPPH free radicals in concentration dependent manner and this activity was highest for this plant. This is because of the presence of higher phenolic contents in this plant. Its medicinal use as a blood tonic and skin infections may be due to its antioxidant activity and high phenolic contents. The Milletia pachycarpa has not been as such subjected for screening of its antioxidant potential however, its methanol extract has been found to be cytotoxic. Out of 14 compound isolated from it, four were found to be cytotoxic in vitro (Ye et al., 2012; Jainul et al., 2013).


Schima wallichii exerted antioxidant effect as indicated by a concentration dependent rise in the neutralization of DPPH radical by chloroform and ethanol extracts. The Schima wallichii has been reported to possess antioxidant activity in FRAP and DPPH assays earlier (Phomkaivon and Areeku, 2009; Subba B. and Paudel, 2014; Sarbadhikary et al., 2015). None of these authors used chloroform and ethanol extracts. However, the magnitude of antioxidant effect observed in the present study was almost similar to them. The medicinal properties of Schima wallichii may be due to the presence of high polyphenolic contents.


The antioxidant activity of Castanopsis indica and Dysoxylum gobara was lesser than all other three plants. The reduced antioxidant activity of these plants may be attributed the presence lower quantity of phenols. The Castanopsis indica has been reported to scavenge superoxide, hydroxyl and ferric free radicals earlier and this activity was attributed to the presence of total phenols (Dolai et al. 2012). Fruit carp of another species Castanopsis tribuloides has been found to scavenge free radicals earlier (Prakash et al., 2012). Reports regarding the antioxidant activity of Dysoxylum gobara are unavailable. However, Dysoxylum gobara has the least antioxidant activity among all the five plants and its total phenolic contents were also the lowest, which explains its low activity.


The antioxidant activity of all five plants may be due to the presence of many polyphenolic compounds that are synthesized by plants for various purposes. We found that the amount total phenolic directly correlates with the DPPH scavenging activity.


5 Conclusions

The investigation of free radical scavenging activity of Milletia pachycarpa, Schima wallichi, Eleagnus caudata, Castanopsis indica and Dysoxylum gobara revealed a concentration dependent elevation in DPPH scavenging activity indicating that these plants possess antioxidant potential. The lower antioxidant activity was observed for Castanopsis indica and the least for Dysoxylum gobara.  This was reflected in the total phenol contents as the plants that had high total phenols showed high antioxidant potential, whereas those that had low total phenols also exhibited lower antioxidant activity. The medicinal property of these plants seems to be directly linked to their total phenolic contents and their ability to scavenge free radicals.


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