Antioxidant activity of leaf extracts of Holoptelea integrifolia (Roxb.) Planch
Or
Antioxidant activity and total Phenolic and Flavonoid content of leaf extracts of Holoptelea integrifolia (Roxb.) Planch
Abstract
Objective: To determine the antioxidant activity, total phenolic and flavonoids contents and their correlation in Holoptelea integrifolia leaves.
Methods: Antioxidant activity was evaluated by 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay. The total phenolic content was determined using Folin-Ciocalteu reagent, whereas Aluminum chloride colorimetric method was used to estimate the flavonoid content.
Results: Methanolic leaf extract showed strongest antioxidant activity (45.63%), followed by pet. ether (40.68%) and chloroform (35.41%) extracts. In contrast, ethanolic extract had lowest antioxidant potential (27.62%). Total phenolic and flavonoids contents in the plant extracts ranged from 45 to 79 mg GAE/g and 5 to 78 mg QE/g, respectively. Those plant extracts with greater free radical scavenging activities also showed the higher content of both phenols and flavonoids, suggesting the positive correlation between polyphenolic content and antioxidant activity.
Conclusions: These findings suggest that total phenolics could be used as an indicator of antioxidant property. H. integrifolia leaves are the potential sources of bioactive phenolic compounds with high antioxidant properties, which can be used as natural antioxidant agents for preventing the progression of many diseases.
Keywords: Polyphenols, Natural antioxidants, free radicals, Holoptelea integrifolia, antioxidant activity
1. Introduction
Endogenous free radicals such as superoxide, nitric oxide and hydroxyl free radicals are known to be the major cause of various chronic and degenerative diseases. Oxidation is a natural process in organisms for the production of energy to fuel biological cycles. Conversely, the uninhibited production of oxygen-derived free radicals is involved in the onset of many diseases such as arthritis, atherosclerosis, rheumatoid and cancer as well as in many degenerative diseases related with ageing (Halliwell and Gutteridge, 1984). In addition, oxidant by-products of normal metabolism cause extensive damage to DNA, proteins, and lipids constituting a major contribution to ageing and also to degenerative diseases of ageing such as cancer, cardiovascular disease, brain dysfunction and cataracts (Diaz et al., 1997; Lang and Lozano, 1998; Metodiewa and Koska, 2000). Although synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertbutylhydroquinone (TBHQ) have been commonly used as antioxidants for years, their safety has long been questioned. Recently synthetic products are being restrained in the industries, because of harmful effects such as human toxicity and carcinogenicity (Oliveira et al., 2008; Oh et al., 2008; Angulo et al., 2009). From this point of view, there is an increasing interest both in the industry and in scientific research towards the use of natural products instead of synthetic products. For thousand years the plant natural products have been used in the medicine without or less harmful effects. Thus, plant extracts appear to be a feasible alternative for this problem and the industries have put the attention in the bioactive phytochemicals present in the plants. Generally, phytochemicals present in the plant extracts are nontoxic, effective at low concentrations, low cost and friendly with the environment. Plant extracts are the complex mixture of several compounds as alcohols, esters, aldehydes, ketones, carbohydrates, terpenes, polyphenols, etc. (Singh et al., 2009). Phenolic compounds are commonly reported to have the best antioxidant activity (Salazar et al., 2008).
In this context, India is a fascinating country for its large endemic plant variety. Crude extracts, purified fractions and pure compounds from several plants of different regions of the country have already been used in an antioxidant approaches and several positive responses have been obtained (Gonzalez et al., 2004).
Ulmaceae is among the plant families that is an important natural resource and provides with many useful products for medicines. Among the plants most used are: Holoptelea integrifolia, Celtis australis, Ulmus parvifolia and Ulmus rubra. Holoptelea integrifolia is the unique source of various types of compounds having diverse chemical structure. The plant is rich in phytochemicals and is well known for its medicinal properties. Bark and leaves are astringent, bitter, antihelmentic, antidiabetic, adaptogenic, antibacterial, antioxidant and are widely used for the treatment of rheumatism, intestinal disorder, leprosy, cutaneous diseases and wound-healing in the form of paste (Sharma et al., 2010; Kumar et al., 2011; Ahmad et al., 2012; Ganie and Yadav, 2014). Paste of the stem bark is externally applied to treat the inflammation of lymph gland, common fever (Singh and Ali, 1994), ringworm and scabies. Stem bark acts as anti-inflammatory agent, especially for eyes (Mudgal and Pal, 1980). Paste of bark and leaf is applied externally for treatment of leucoderma (Benjamin and Christopher, 2009; Mahmud et al., 2010).
Given that the H. integrifolia possess many useful biological activities, the present study was undertaken to measure Phenolic and Flavonoid contents of leaf extracts and to evaluate their antioxidant activity to exploit their therapeutic utilities to combat various diseases.
2. Materials and Methods
2.1. Chemicals
Quercetin, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and gallic acid were purchased from Sigma-Aldrich, USA. Folin- Ciocalteu reagent was obtained from Merck, Germany. All other chemicals and solvents used in the study were of analytical grade procured locally.
2.2. Plant material
Fresh leaves of H. integrifolia were collected from different areas of Rohtak, Haryana, India. The collected leaves were transported to the department of Botany, Maharshi Dayanand University, Rohtak (Haryana). Immediately the samples were washed thoroughly and shade dried. The dry samples were ground in a mixer-grinder. The fine and dried powder was stored in black plastic bags at room temperature.
2.3. Preparation of plant extracts
Soxhlet extraction method following Okeke et al. (2001) was used for extraction of H. integrifolia leaves.
A known amount of leaf powder (50g) was extracted separately with different solvents viz., . Powder was packed in extraction thimble and placed in an extraction chamber which was suspended above the flask containing the solvent. The flask was heated and the solvent evaporated and moved into the condenser where it was converted into a liquid that trickled into the extraction chamber containing the plant material. The extraction chamber was designed so that when the solvent surrounding the sample exceeded a certain level, it overflowed and trickled back down into the boiling flask. The extraction was done till the dark colour of the seeds turns colourless. At the end of the extraction process, the flask containing the plant extract was removed and solvent was evaporated using a rotary evaporator. The crude extracts were stored at 4 OC in a dark place before their use in the antioxidant assay and quantification of total phenolic and flavonoid contents.
2.4. Determination of DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging activity
First, the antioxidant activity in the extracts was evaluated as the DPPH free radical-scavenging activity. This assay was determined following Choi et al. (2002) with some minor modifications.
The method depends on the reduction of purple DPPH (Sigma- Aldrich) to a yellow colored diphenyl picrylhydrazine. Each sample stock solution (10 mg/ml) was diluted to final concentrations of 9, 8, 7, 6, 5, 4, 3, 2 and 1 mg/ml. One ml of a 0.3 mM DPPH solution was added to 2.5 ml of sample solution of different concentrations. Ascorbic acid (HiMedia) was used as positive control. As DPPH is sensitive to light, it is exposed to the minimum possible light. These solutions were allowed to react at room temperature for 30 minutes. The absorbance values were measured at 518 nm and converted into the percentage antioxidant activity using the following equation:
Scavenging capacity (%) = Abs control – Abs sample / Abs control× 100
Where Abs is the absorbance
2.5. Determination of Total Phenolic Content (TPC)
Total phenolic content (TPC) in the extracts was determined using the Folin-Ciocalteu reagent method (Singleton et al., 1999; McDonald et al., 2001; Liu et al., 2008). This method depends on the reduction of FCR by phenols to a mixture of blue oxides which have a maximal absorption in the region of 765 nm. A stock solution of plant extract was prepared to the concentration of 10 mg/ml. To 1.0 ml of extract, 5 ml of Folin-Ciocalteu reagent was added. The solution was vortexed and incubated in the dark for 3 minutes. After that 5 ml of sodium carbonate (75 g/l) solution was added to the mixture and mixed thoroughly. The mixture was incubated in the dark for 1 hour. The absorbance was read at 765 nm. Blank consisted of 5 ml Folin-Ciocalteu reagent, 1 ml solvent and 4 ml sodium carbonate solution. The same procedure was repeated for the standard solution of gallic acid and the calibration line was construed. The samples were prepared in triplicate for each analysis and the mean value of absorbance was recorded. Based on the measured absorbance, the concentration of phenolics was read (mg/ml) from the calibration line; then the total content of phenolic compounds were expressed as gallic acid equivalents (GAE), calculated by the following formula:
C = c × V/m
Where,
C – total content of phenolic compounds (mg of GA/g of extract)
c – the concentration of gallic acid established from the calibration curve (mg/ml)
V- the volume of extract (ml)
m – the weight of pure plant extract (g)
2.6. Estimation of flavonoid Content
Flavonoid content of each extsract was determined by aluminum chloride method (Chang et al., 2002; Ebrahimzadeh et al., 2008; Madaan et al., 2011) with some minor modifications. Quercetin was used as a standard to construct the calibration curve. Briefly, 0.5 ml of diluted standard quercetin solutions (20, 40, 60, 80 and 100 µg/ml) were separately mixed with 1.5 ml of solvent, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 30 min., the absorbance of the reaction mixture was measured at 415 nm. Similarly, 0.5 ml of each plant extracts were reacted with aluminum chloride for the determination of flavonoid content as described above. The flavonoid content was calculated as mean±SD (n=3) and expressed as mg/g of quercetin equivalent (QE) of dry extract.
2.7. Data analysis
The results are expressed as mean±SD. Karl Pearson’s correlation coefficient (r) was used to analyze the level of correlation between antioxidant activity and phenolic and flavonoid contents. Oneway ANOVA was used to analyze level of statistical significance between phenolic and flavonoid contents.
3. Results
3.1. DPPH Radical-Scavenging Activity
The antioxidant activity of four different extracts from H. integrifolia leaves was determined using DPPH radical scavenging assay. The activity is expressed in terms of percentage of DPPH radical inhibition. Parallel to examination of the antioxidant activity of plant extracts, the values for ascorbic acid, as a standard compound, were obtained and compared to the values of the plant extracts. All the four leaf extracts displayed striking DPPH radical scavenging activities. The obtained values ranged from 38.52 to 45.63% in methanolic extract (Fig. ),30.39 to 40.68% in pet. ether extract (Fig. ), 22.25 to 35.41% in chloroform extract () and 18.02 to 27.62% in ethanolic extract (Fig. ). The methanolic extract (10 mg/ml), showed the highest radical-scavenging activity (45.63%). The pet. ether and chloroform leaf extracts showed moderate, while ethanolic extract had lowest antioxidant activity. The radical scavenging potential of the extracts was dose-dependent. Compared to the plant extracts, the standard ascorbic acid showed more powerful antioxidant activity, ranged from 28 to 60%.
3.2. Total Phenolic and Flavonoid Contents
Total phenolic and flavonoids contents of the various leaf extracts of H. integrifolia are expressed in terms of GAE and QE, respectively and presented in Table 1. Results showed that the different extracts contained variable amounts of these compounds. Methanolic extract was found to contain the highest amount of phenolic compounds (79.0±2.5 mg GAE/g), followed by pet. ether, chloroform and ethanolic extracts. Among four organic extracts, maximum amount of flavonoid content (80.0±2.0 mg QE/g) was found in pet. ether, followed by methanolic, chloroform and ethanolic extracts. A significant difference in the phenolic and flavonoid contents of different extracts was observed by ANOVA single factor test.
Table 1: Total Phenolic and Flavonoid contents of leaf extracts of H. integrifolia
Extract Total Phenolic Content Flavonoid Content
(mg GAE/g) (mg QE/g)
Methanolic 79.0±2.5 78.0±1.73
Pet. Ether 78.0±1.5 80.0±2.0
Chloroform 60.0±1.73 68.0±2.17
Ethanolic 45.0±3.27 5.0±0.50
All values are expressed as Mean ± SD of three replicates
3.3. Correlation between Phenolic and Flavonoid Contents and antioxidant activity
Based on correlation analysis, DPPH radical-scavenging activity of H. integrifolia leaf extracts was strongly correlated with total phenolic content (r = 0.969) though moderately correlated with flavonoid content (r = 0.894).
4. Discussion
Various methods are used to investigate the antioxidant property of samples (diets, plant crude extracts, isolated compounds, commercial antioxidants etc.). In the present study, we employed DPPH assay to determine the antioxidant potential of H. integrifolia leaf extracts. The model of scavenging the stable DPPH radical is widely used to evaluate the free radical scavenging ability of wide range of samples (Lee et al., 2003). The reduction of the DPPH by the process of either proton or electron donation can be monitored spectrophotometrically, as the DPPH upon reduction changes color from violet to yellow. Therefore, the substances capable of reducing DPPH could be considered as an antioxidant and radical scavengers (Kris-Etherton et al., 2002). In the present study, marked variations were observed in the DPPH radical scavenging activities of H. integrifolia leaf extracts. Methanolic leaf extract showed best free radical scavenging activity than other extracts. These differences in the results can be attributed at solvent used in the study as chloroform extracts certain compounds of low or no polarity, while, methanol is a solvent with higher polarity. The extraction of the phytocompounds largely depends on the solvent used, time and temperature of extraction as well as the chemical nature of the sample (Shimada et al., 1992).
Our findings on the antioxidant activity are in agreement with the previous report of Srivastava et al. (2013) from aqueous stem bark extract of H. integrifolia. They reported that DPPH radical scavenging activity ranged from 13.14 to 55.17%, comparable to standard ascorbic acid (22.56–93.68%).
Numerous reports are available, which demonstrated the usefulness of phenolic and flavonoid compounds in biological activities such as antioxidant, antidiabetic, hepatoprotective, anti-inflammatory, antimicrobial, anticancer etc. (Sulaiman et al., 2011; Kumar and Pandey, 2013). The antioxidant activity of phenolic compounds is mainly due to their reduced properties which allow them to act as metal chelators, absorb and neutralize free radicals (Mishra et al., 2010). Flavonoids and tannins are considered to be the most promising polyphenolic compounds among plant secondary metabolites (Tomczyk et al., 2010). Antioxidant activity of flavonoids depends on the structure and substitution pattern of hydroxyl groups (Sharififar et al., 2008). Therefore, the total phenolic and flavonoid contents of different extracts of H. integrifolia leaves and their relation with the antioxidant activity were estimated. Phenols and polyphenolic compounds, such as flavonoids, are widely found in food products derived from plant sources, and they have been shown to possess significant antioxidant activities (van Acker et al., 1996). .
The correlation between total phenolics and antioxidant activity has been widely studied in different foodstuffs such as fruit and vegetables (Klimczak et al., 2007; Kiselova et al., 2006; Jayaprakasha et al., 2008; Kedage et al., 2007). As reported, antioxidant activity of fruits and vegetables significantly increases with the presence of high concentration of total polyphenol content. In the present study, the correlation between total phenolic and flavonoids contents and radical scavenging activity of H. integrifolia leaf extracts were analyzed. In general, extracts with a high radical scavenging activity showed a high phenolic and flavonoid contents as well. One previously published report on Holoptelea integrifolia revealed that the higher antioxidant activity of methanolic stem bark extract might be due to the higher phenolic content of MSBE (Reddy et al., 2008).
It is interesting to note that despite of that H. integrifolia ethanolic extract had lower TFC value respect to methanolic, chloroform and pet. ether extracts, it showed a good antioxidant activity. It may be due to flavonoid compounds are not the unique phytochemicals to possess antioxidant properties (Choe and Min, 2009). Furthermore, the extracts are very complex mixtures of many different compounds with distinct activities (Mensor et al., 2001; Hou et al., 2003). This lack of relationship is in agreement with other literature (Heinonen et al., 1998; Anagnostopoulou et al., 2006; Nickavar et al., 2007).
5. Conclusions
The leaf extracts of H. integrifolia upon quantification were found to be rich in phenolic compounds (phenols and flavonoids) and therefore exhibited potential scavenging activity against DPPH. The better extraction of total phenolics was clearly associated to the antioxidant capacity. Methanolic extract was the best source for the antioxidant activity in our study. Generally, we can suggest that the plant extracts used in the study are a natural source of bioactive compounds and of great value that could be used properly in the healthcare and pharmaceutical industries. However, subsequent studies like in vitro and in vivo correlation studies along with isolation of compounds, fractionation of the extract and characterization of the structures are necessary to confirm novel treatment strategies for free radical induced diseases.
Conflict of interest
We declare that we have no conflict of interest.
Acknowledgments
Financial assistance from Haryana Council for Science and Technology (HSCST), Panchkula and University Grants Commission (UGC), New Delhi is thankfully acknowledged.
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