INTRODUCTION:

Oxidative stress plays an important role in the progression of many diseases like asthma. Many endogenous factors like reactive oxygen species (ROS) and exogenous factors like pollution are responsible for oxidative stress.^[1]Asthma is a chronic obstructive disorder caused by inflammation of the airways and oxidative stress is believed to be involved in its pathogenesis.^[2] Alleviated oxidative stress contributes to the progression of airway inflammation and release of various pro-inflammatory mediators^[3-7] which are responsible for causing severe asthma.^[8-9]

Previous studies have shown there exists a relationship between increased levels of reactive oxygen species and pathogenesis of asthma.^[2] Also, in a study it was seen that there was an increase in the incidence of asthma where the intake of food containing antioxidants was low.^[10-12] Therefore, an effective way to manage bronchial asthma is to control oxidative stress.^[2]

Antioxidants have the property to scavenge free radicals and protect cells against oxidative damage. Cellular damage occurs when there is an imbalance between antioxidants and reactive oxygen species in the body. The antioxidants that protect cells in the body are GSH, catalase, superoxide dismutase etc.^[13]Synthetic antioxidants possess numerous side effects.^[14] Therefore, scientists are in search of natural antioxidants derived from plants, which do not have health hazards.^[15] The natural antioxidants stimulate endogenous antioxidants to neutralize oxidative stress. The secondary metabolites like flavonoids, alkaloids, terpenoids, phenolics etc found in plants have shown various pharmacological properties and many of them have been evaluated for their antioxidant property.^[16]

Flavonoids are polyphenolic compounds which have shown to exhibit free radical scavenging activity.^[17-18]Phenolics from plants are becoming important due to their excellent antioxidant properties.^[19]These secondary metabolites are known to possess many other biological activities as well.^[20-21]

Mangifera indica is a fast growing tree found in all parts of the country. Many pharmacological activities have been reported on various parts of this tree.^[22]

The leaves of Nicotiana tabacum have been used in Indian traditional medicine for years. Many pharmacological activities are also reported on this plant.^[23]

The present study is designed to investigate and compare the total phenolic content, total flavonoid content and antioxidant potential of aqueous ethanolic extract of leaves of Mangifera indica and Nicotiana tabacum using two invitro methods namely, DPPH (2,2- Diphenyl-1-picrylhydrazyl) assay and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay.

MATERIALS AND METHODS:

Chemicals:

The chemicals used in this study were purchased from various suppliers. Gallic acid, Sodium hydroxide, DPPH (2,2-diphenyl-1- picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were purchased from Sigma-Aldrich. Folin-ciocalteu, Sodium carbonate, Aluminium chloride, Quercetin, Sodium nitrite and potassium persulfate were purchased from Merck.

Preparation of Crude Extracts:

The leaves of Mangifera indica and Nicotiana tabacumwere collected from the local regions of Bilaspur, Chhattisgarh in the months of January to March. The leaves were washed with distilled water to remove the undesirable material and dried under shade for three weeks. The dried material was ground to coarse powder and stored in air tight containers for further use. Cold percolation technique was used for exhaustive extraction of the leaves. For this, 500 g of powered material was extracted with 1000 ml of aqueous ethanol (1:1) for 72 hours with subsequent filtration. The extract was filtered using a Whatmann filter paper no. 1. The filtrate obtained was evaporated to dryness using a rotary evaporator (BUCHI, Switzerland) at 40°C temperature under reduced pressure. It rendered a viscous concentrate of dark green colour. Then the crude extracts were dried in freeze drier and preserved at +4°C.

DETERMINATION OF TOTAL PHENOLIC CONTENT:

The total phenolic content in the plant extract was estimated using Folin-Ciocalteu Method.^[24]Standard solution of Gallic Acid was prepared in the range of 100 µg/ml – 500 µg/ml. Reaction mixture consisted of an aliquot (1ml) of plant extract/ gallic acid and 10 ml of distilled water in a volumetric flask of 25 ml. 1 ml of Folin-Ciocalteu Reagent was added to it and the flask was shaken and kept aside for 5 min. Than 10 ml of 7.5% Sodium Carbonate solution was added and the total volume was made upto the mark with distilled water. The solution was kept for incubation at room temperature for 2 hrs. A blank was prepared using methanol. All the experiments were carried out in triplicates. After incubation the colour of the reaction mixture turned deep blue. The absorbance of extract/ gallic acid/ blank was measured at 750 nm using UV-Vis spectrophotometer(Shimadzu, USA). A calibration curve of the standard was plotted and the total phenolic content was calculated from it. The results of the total phenolic content in the extracts was expressed as mg of GAE/ gm DW.

DETERMINATION OF TOTAL FLAVONOID CONTENT:

The total Flavonoid Content in the plant extract was evaluated using Aluminium Chloride Colorimetric assay.^[25] Standard solution of Quercetin was prepared and five serial dilutions were made to obtain a concentration of 100-500 µg/ml. In a 10 ml volumetric flask containing 1 ml of standard solution of quercetin or plant extract (1mg/ml) and 4 ml of distilled water were added. To this 0.5 ml of 5 % sodium nitrite solution was added and the reaction mixture was incubated at room temperature for 10 minute. After 10 minutes 0.5 ml of 10 % aluminium chloride solution and 2 ml of 1 M sodium hydroxide was added. Finally the volume was made up with distilled water. The solution was allowed to stand for 15 min for the reaction to complete. An orange colour was developed. The Absorbance was measured against blank at 510 nm. All the experiments were carried out in triplicates. A calibration curve of Quercetin was plotted and the total flavonoid content of the extract was calculated from line of regression and expressed as mg QE/g DW.

DPPH FREE RADICAL SCAVENGING ASSAY:

The antioxidant activity of the aqueous ethanolic extracts of Mangifera indica and Nicotiana tabacum were assessed using the stable 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH).^[26]Methanolic solution of DPPH is purple in colour. Some phytochemicals present in plants have the ability to donate hydrogen atom and show free radical scavenging property. This antioxidant activity causes decolourisation of DPPH solution. Hence, with an increase in the concentration of antioxidants in plants, the absorbance will decrease. 0.004% (w/v) solution of DPPH in methanol was freshly prepared and kept aside to protect from light. The extract solution was prepared at a concentration of 1 mg/ ml. Five serial dilutions of the plant extracts were prepared to give concentrations of 100-500 μg/ml. Ascorbic acid was used as the standard. For the assay, 3 ml of DPPH solution was mixed with 5 ml of extract/ ascorbic acid. The solutions were allowed to stand for 60 min and the absorbance was measured at 517 nm at a UV Spectrophotometer. 2 ml of DPPH 0.004% (w/v) and 1 ml of methanol was used as control. All the determinations were carried out in triplicate.

The percentage of radical scavenging by the samples was calculated using the formula ^[27] Inhibition (%) = [{Abs control - Abs sample}/Abs control] × 100 where, Abs control is the absorbance of the DPPH radical (0.004% w/v) +methanol, Abs sample is the absorbance of DPPH radical + extract/standard. The IC₅₀values were calculated from the calibration curve using linear regression. IC₅₀is the concentration of extract which produces 50 % inhibition. A low value for IC₅₀indicates higher antioxidant potential. The results were expressed as percentage DPPH free radical scavenging potential and IC₅₀ values of the extracts.

ABTS⁺RADICAL CATION DECOLORIZATION ASSAY:

The ABTS free radical-scavenging activity of the extracts was determined by ABTS radical cation decoloration assay.^[28] Stock solutions of ABTS (7 mM) and potassium persulfate (140 mM) were prepared and stored in amber coloured bottles. 5 ml of ABTS solution was mixed with 88 µl potassium persulfate to produce ABTS⁺ free radical cation. The solution mixture was kept in dark for 16 h for the reaction to complete. Before using the ABTS⁺ free radical solution, 1 ml of it was diluted with about 100 ml of 80% methanol so as to obtain an absorbance of 0.700 ± 0.005 at 734 nm. 0.2 mL of the extract at various concentrations was taken in different test tubes and 2.8 ml ABTS⁺ radical solution was added to it. The reaction mixture was kept at room temperature for 30 min and the absorbance was recorded at 734 nm. Ascorbic acid was used as the standard. A calibration curve of absorbance vs concentration was plotted. The percentage inhibition of ABTS free radical was calculated from the following formula:

Radical Scavenging Power (%) = [(Ac − As)/Ac] × 100

where Ac and As are the absorbances of the control and sample, respectively.ABTS free radical cation solution was used as control.

IC₅₀ values of the extracts were calculated from the standard graph using regression analysis as done in the previous assay. The results were expressed as percentage scavenging ability and IC₅₀ values of the extracts.

STATISTICAL ANALYSIS:

All the experiments were carried out in triplicates. The result was presented as mean ± standard deviation. One-way ANOVA followed by Tukey’s test was used for analysis of the datausingGraphPad Prism (version 7.03) and Microsoft Excel 2007.p value< 0.05 was regarded as significant.

RESULTS:

The aqueous ethanolic extracts of leaves of Mangifera indica and Nicotiana tabacum were obtained using cold percolation method. It is seen that various substances exhibit antioxidant activity through various mechanisms. Therefore, it is always better to use two or more than two methods to study the antioxidant activity. In the present study, the antioxidant property of the extracts were determined using two in vitro assays, DPPH free radical scavenging assay and ABTS⁺ radical cation decolorization assay. Extracts were also analyzed for their phenolic and flavonoid contents. Table 1 shows percentage yield of the extracts.

TOTAL PHENOLIC AND FLAVONOID CONTENT:

The total phenolic and flavonoid contents of the aqueous ethanolic extracts of Mangifera indica and Nicotiana tabacum are shown in figure 1 and 2, respectively. Results for the total phenolic content and total flavonoid content of the extracts were expressed as mg GAE/ g DW and mg QE/g DW. Calibration curve of the standard, gallic acid and quercetin are shown in figure 3 and 4. Total phenolic content and total flavonoid content of the extract of Mangifera indica were (67.72 ± 0.02) mg GAE/g DW and (50.16 ± 0.03) mg QE/g DW, respectively. Whereas, total phenolic content and total flavonoid content of the extract of Nicotiana tabacum were (39.54 ± 0.03) mg GAE/g DW and (31.96 ± 0.02) mg QE/g DW, respectively. The results show that the total phenolic and total flavonoid content in extract of Mangifera indica was higher than in Nicotiana tabacum.

DPPH FREE RADICAL SCAVENGING ASSAY:

Figure 5 compares the scavenging abilities of the aqueous ethanolic extracts of Mangifera indica, Nicotiana tabacum and the standard, ascorbic acid. The plants showed their ability to scavenge DPPH free radicals in a dose-dependent manner. At various doses the extract of Mangifera indica exhibited significantly (p<0.05) higher antioxidant activity than the extract of Nicotiana tabacum. The results from the experiment shows that the DPPH free radical scavenging ability of the extract of Mangifera indica was comparable to that of the standard, ascorbic acid (p>0.05). At the highest concentration of 500 μg/ml the percentage scavenging activity of Mangifera indica was 73.56 ± 0.01 as compared to Nicotiana tabacum which was only 40.22 ± 0.01. At the same concentration the standard showed 87.35 ± 0.01 percent scavenging activity, which was comparable to Mangifera indica. The significant (p<0.05)difference between Mangifera indica and Nicotiana tabacum in their ability to quench DPPH radicals could be due to the presence of various secondary metabolites and higher concentrations of phenolics and flavonoids in the aqueous ethanolic extract of Mangifera indica. Table 2 summarises the DPPH free radical scavenging ability of the two extracts. Figure 6 compares the IC₅₀ of the extracts. The IC₅₀ of ascorbic acid was 42.11 ± 0.01 μg/ml whereas the IC₅₀ of the extracts of Mangifera indicaand Nicotiana tabacumwere 192.64 ± 0.01 and 693.67 ± 0.01 μg/ml, respectively. The free radical scavenging activity of the two extracts and the standard was in the following order ascorbic acid >Mangifera indica>Nicotiana tabacum.

ABTS⁺RADICAL CATION DECOLOURATION ASSAY:

The ABTS⁺radical cation scavenging ability is a useful method in determining the antioxidant potential of a sample. The percentage scavenging power of the extracts of Mangifera indica and Nicotiana tabacum on ABTS⁺ is shown in Table 3. From statistical analysis it was found that a significant (p<0.05)difference existed between the extracts ofMangifera indica and Nicotiana tabacum in their ability to scavenge ABTS⁺radicals. In the assay, the absorbance at 734 nm decreased with an increase in the concentration of the extracts/ standard. Greater the decolouration of the ABTS⁺, higher is the radical scavenging power of the sample and it is due to the ability of the sample to donate protons (presence of phenolic compounds). ABTS⁺ radical cation scavenging ability increases with the increase in the percentage of ABTS⁺radical cation inhibition. Both the extracts showed a decrease in absorbance with increase in their concentration indicating the ability to scavenge ABTS⁺radical cation in a dose dependent manner. At the highest concentration of 500 μg/ml, the percentage ABTS⁺ radical scavenging activity of Mangifera indicaand Nicotiana tabacumwas 62.85±02 and 35.71 ± 0.03, respectively. Standard, ascorbic acidat a concentration of 500 μg/ml showed 78.57 ± 0.02 percent ABTS⁺ radical scavenging power. The data shows there exists a significant (p<0.05) difference between the standard and Nicotiana tabacum as well as between the two plants in their power to scavenge ABTS⁺free radicals.Mangifera indica possessed greater power to scavenge the ABTS⁺radicals. Figure 7 shows the ABTS⁺ Radical Cation Scavenging Power of the extracts and ascorbic acid. The IC₅₀ of extracts of Mangifera indica, Nicotiana tabacum and ascorbic acid were100.14 ± 0.01, 278.66 ± 0.02 and 865.60 ± 0.02 μg/ml, respectively,(Figure 8 ).

Table 1. Percentage Yield of the extracts of Mangifera indica and Nicotiana tabacum

Extract	Color	Odour	% Yield (w/w)
Mangifera indica	Dark green	Odourless	18.24 %
Nicotiana tabacum	Dark green	Characteristic	16.51 %

Table 2: DPPH free radical scavenging activity of extracts of Mangifera indica and Nicotiana tabacum

Concentration of Extracts (μg/ml)	DPPH Free Radical Scavenging Activity
Concentration of Extracts (μg/ml)	Mangifera indica	Nicotiana tabacum
100	42.52 ± 0.01	19.54 ± 0.01
200	50.57± 0.02	24.13 ± 0.01
300	58.62 ± 0.02	29.88 ± 0.01
400	66.66 ± 0.02	34.48 ± 0.02
500	73.56 ± 0.02	40.22 ± 0.01

Table 3: ABTS⁺ radicalscavenging power of extracts of Mangifera indica and Nicotiana tabacum

Concentration of Extracts (μg/ml)	ABTS⁺ Radical Cation Scavenging Activity
Concentration of Extracts (μg/ml)	Mangifera indica	Nicotiana tabacum
100	38.57 ± 0.01	12.87 ± 0.01
200	45.71 ± 0.01	18.57 ± 0.02
300	52.85 ± 0.02	21.42 ± 0.02
400	58.57 ± 0.02	28.57 ± 0.03
500	62.85 ± 0.02	35.71 ± 0.03

Figure 1. Total Phenolic Content expressed as mg GAE/ gm DW in plant extracts. Data are expressed as mean ± SD (n=3)

Figure 2. Total Flavonoid Content expressed as mg QE/ gm DW in plant extracts. Data are expressed as mean ± SD (n=3)

Figure 3. Calibration curve of Gallic acid

Figure 4. Calibration curve of Quercetin

Figure 5: DPPH Free Radical Scavenging ability of the extracts and ascorbic acid

Experiments were performed in triplicate. Data are expressed as mean±SD (n=3)

Figure 6. IC₅₀ of the extracts and ascorbic acid for DPPH free radical scavenging assay

Experiments were performed in triplicate. Data are expressed as mean±SD (n=3)

Figure 7: ABTS⁺ Radical Cation Scavenging Power of the extracts and ascorbic acid

Experiments were performed in triplicate. Data are expressed as mean±SD (n=3)

Figure 8. IC₅₀ of the extracts and ascorbic acid for ABTS radical cation scavenging power

Experiments were performed in triplicate. Data are expressed as mean±SD (n=3)

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Received on 06.10.2017 Modified on 08.11.2017

Research J. Pharm. and Tech 2018; 11(2):717-722.

DOI: 10.5958/0974-360X.2018.00135.X