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Research Article
Journal of Chinese Integrative Medicine: Volume 13   July, 2015   Number 4

DOI: 10.1016/S2095-4964(15)60183-2
Comparative and quantitative analysis of antioxidant and scavenging potential of Indigofera tinctoria Linn. extracts
1. Rashmi Singh (Department of Bioscience and Biotechnology, Banasthali University, Rajasthan-304022, India )
2. Shatruhan Sharma (Maa Aananad Mai Institute, Jaipur-12, Rajasthan-304022, India )
3. Veena Sharma (Department of Bioscience and Biotechnology, Banasthali University, Rajasthan-304022, India )

ABSTRACT

OBJECTIVE: To compare and elucidate the antioxidant efficacy of ethanolic and hydroethanolic extracts of Indigofera tinctoria Linn. (Fabaceae family).

METHODS: Various in-vitro antioxidant assays and free radical-scavenging assays were done. Quantitative measurements of various phytoconstituents, reductive abilities and chelating potential were carried out along with standard compounds. Half inhibitory concentration (IC50) values for ethanol and hydroethanol extracts were analyzed and compared with respective standards.

RESULTS: Hydroethanolic extracts showed considerably more potent antioxidant activity in comparison to ethanol extracts. Hydroethanolic extracts had lower IC50 values than ethanol extracts in the case of DPPH, metal chelation and hydroxyl radical-scavenging capacity (829, 659 and 26.7 μg/mL) but had slightly higher values than ethanol in case of SO2- and NO2-scavenging activity (P < 0.001 vs standard). Quantitative measurements also showed that the abundance of phenolic and flavonoid bioactive phytoconstituents were significantly (P < 0.001) greater in hydroethanol extracts (212.920 and 149.770 mg GAE and rutin/g of plant extract respectively) than in ethanol extracts (211.691 and 132.603 mg GAE and rutin/g of plant extract respectively). Karl Pearson’s correlation analysis (r2) between various antioxidant parameters and bioactive components also associated the antioxidant potential of I. tinctoria with various phytoconstituents, especially phenolics, flavonoids, saponins and tannins.

CONCLUSION: This study may be helpful to draw the attention of researchers towards the hydroethanol extracts of I. tinctoria, which has a high yield, and great prospects in herbal industries to produce inexpensive and powerful herbal products.

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Citation: Singh R, Sharma S, Sharma V. Comparative and quantitative analysis of antioxidant and scavenging potential of Indigofera tinctoria Linn. extracts. J Integr Med. 2015; 13(4): 269–278.

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1 Introduction

In recent years, the incidences of diseases like cancer[1], high blood pressure and heart-related troubles[2] have been increasing with greater risk of adverse outcomes. Antioxidants may provide some protection against these health conditions, which are often caused by environmental exposure[3,4]. Herbs are an excellent source of various phytoconstituents, such as flavonoids, polyphenols, tocopherols, tocotrienols, ascorbic acid and carotenoids, which account for their antioxidant activity[5]. Medicinal plants can possess very powerful pharmacological properties. Phytomedicines and isolated bioactive compounds have been reported to have a wide spectrum of biological functions and many have been used as medicines in folklore for treating a similarly wide range of diseases[6].
Indigofera is a genus that contains approximately 700 species, which are distributed across various tropical regions. Indigofera tinctoria Linn. (true indigo; Fabaceae family) is distributed throughout south East Africa, Tropical Africa as well as Tropical America. This plant is cultivated in Southern India, especially Tamil-Nadu[7]. It is a deciduous shrub, reaching 1–2 m in height, which may be annual, biennial or perennial. The indigo dye obtained from this plant has been traded internationally since ancient times. I. tinctoria is a nitrogen-fixing legume that increases soil fertility. Indigotin, a colorless glycoside responsible for the blue color dye, has antiseptic and astringent properties[8]. This plant is traditionally used in the treatment of health problems, such as constipation, liver disease, heart palpitation and gout[9]. It is also used in naturopathic medicine to treat splenomegaly, echolalia, chronic bronchitis, asthma[10], ulcers, skin diseases, diuretic and hair growth problems[11]. This plant also possesses anti-toxic[12] and antimicrobial properties[13–15]. Further, it has been reported that the decoction of I. tinctoria leaves was used in the treatment of venomous insect bites, burns and scalds[16].
The present study is aimed at quantifying the bioactive compounds and antioxidant activities of ethanolic and hydroethanolic extracts of I. tinctoria. The results clearly point out its valuable aspect in future as a potential phytomedicine.

 
  

2 Materials and methods

2.1 Plant material and authentication
Shade dried I. tinctoria Linn., collected from Tindivanam, Tamilnadu, India, was purchased from S.M. Heena Industries, Jaipur, Rajasthan (Order No. and date: 01/19-06-2012; Batch No. 26). Plant sample was identified, submitted and authenticated at Harbarium Section of Department of Bioscience and Biotechnology, Banasthali University (Auth entication number-BURI-13515).
2.2 Extract preparation
Dried plant material was minced in a grinder (Model UNOVA, mfd. by VISHVA Enterprises, Mumbai, 18 000 × g), sieved (Sethi Standard Test Seive, Yamberzal International, Jaipur; width of aperture 1 mm, British standard sieve mesh series = 16) and extracted with Soxhlet[17] using a step gradient of nonpolar to polar solvents (petroleum ether, chloroform, ethyl acetate and ethanol) to get ethanolic extracts. Hydroethanolic extract was prepared with 80% ethanol after defatting with ether. Ethanolic extract (EtOH) and hydroethanolic extract (HE) were used for in-vitro studies. The extracts were filtered and dried under reduced pressure in a rotary evaporator (Heidolph, Incarp Instruments Pvt. Ltd., Germany) to afford 94.5 g and 258.25 g/kg plant powders respectively.
2.3 Chemicals
Rutin trihydrate, vanillin, plant saponin, 2-deoxyribose, nitroblue tetrazolium chloride (NBT), reduced-nicoinamide adinine dinucleotide (NADH), trichloroacetic acid (TCA), quercetin, 2,2′-bipyridyl, tannic acid (pure), sulphanillic acid, N-(1-naphthyl) ethylenediamine dihydrochloride and thiobarbituric acid (TBA) were purchased from Himedia, Mumbai. Folin-ciocalteu’s reagent (FCR), ascorbic acid and hydrogen peroxide were purchased from Merck, USA. 2,4,6-Tri(2-pyridyl)-5-triazine (TPTZ), FeSO4, gallic acid and phenazine methosulfate (PMS) were purchased from Sisco Research Laboratory (SRL), Mumbai. Ethylene diamine tetraacetic acid (EDTA), potassium ferricyanide and ammonium molybdate were purchased from Qualigens, Mumbai, India whereas 2,2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma Aldrich, USA. All other unmentioned chemicals and reagents were purchased from other firms like S.D. Fine Chemicals, Central Drug House, Mumbai, Ranbaxy (RFCL) and Fisher Scientific. All chemicals mentioned above were of analytical grade and of highest purity (98%–99%).
2.4 Quantitative measurements of various bioactive compounds
2.4.1 Total phenolic content
Total phenolic content (TPC) was determined[18] by using FCR, which contains phosphotungstate metal. Phenols present in the plant sample react with this metal and change the color of the solution to blue, the absorbance of which is directly proportional to the TPC in sample. Different extract dilutions (0.05–0.65 mg/mL) in respective solvents along with gallic acid standards were mixed with 1 mL diluted FCR and left for 5 min. This was followed by addition of 7% Na2CO3 and distilled water. The contents were mixed well and incubated in dark for 90 min. Subsequently samples were centrifuged at 10 000 × g for 5 min (Remi, Mumbai, India, model C-24BL; Serial # LACI-10387) to remove precipitate before the absorbance was measured at 750 nm (Systronics-2080, double beam, UV-VIS spectrophotometer, Japan) against the sample blank.
2.4.2 Total flavonoid content
Total flavonoid content (TFC) in plant samples was determined following the protocol of Jia et al[19]. To dilute samples of plant extracts in respective solvent (0.05–0.65 mg/mL) and standard rutin (1 mg/mL), 2 mL distilled water and 0.15 mL of 5% NaNO2 were added. After 6 min, 10% AlCl3 was added and incubated for 6 min. Thereafter, distilled water was added to make a final volume of 5 mL, and allowed to stand for 15 min. A pink-colored complex was formed in the presence of flavonoids, and its absorbance was measured at 510 nm against the sample blank.
2.4.3 Total saponin content
Total saponin content (TSC) in I. tinctoria extracts was measured using the vanillin-perchloric method with slight modification[20]. To different diluted samples of plant extracts in respective solvent (0.05–0.65 mg/mL), as well as with reference plant saponin, 400 μL of vanillin-acetic acid solution (5%, w/v) and 1.6 mL perchloric acid were added and heated to 70–75 ℃ for 15 min. Later this solution was cooled on ice for 1–2 min and then, 2.5 mL of glacial acetic acid was added. The solution was well mixed and the absorbance was measured at 550 nm against the sample blank.
2.4.4 Total tannin content
Total tannin content (TTC) was determined following a FAO/IAEA protocol[21], in which 1 mL of different dilutions of plant extracts (0.05–0.65 mg/mL) and tannic acid standard were mixed with 8 mL of double-distilled water, diluted FCR and 20% Na2CO3. The absorbance of this solution was measured at 775 nm against the sample blank.
2.4.5 Total proanthocyanidine content
Total proanthocyanidine content in I. tinctoria extracts were evaluated following the protocol of Hiermann et al[22] in which different concentrations (0.05–0.65 mg/mL) of samples and rutin standard were mixed with 1 mL of 70% ethanol, 25% HCl and 5% distilled water before incubation at 85–90 ℃ in a water bath (Sonar, ISO-9001-2000, New Delhi, India) for 80 min. After cooling, n-butanol (1.5 mL) was added, and the absorbance of yellow-pink-colored complex was measured at 545 nm.
2.4.6 Calculation of concentrations of phytoconstituents
All results were calculated[23] and expressed as milligram standard equivalents/gram of dry weight of plant material using formula:
C = c·V/m
where, C = content in plant extract (mg/g); c = content of phytoconstituents in diluted plant sample that comes from standard calibration curve (mg/mL); V = volume of sample (mL).
y = mx + c
where x = absorbance and y = standard equivalent; m = weight of plant extracts (g).
2.5 In-vitro free radical and antioxidant potential
2.5.1 Free radical-scavenging activity
2.5.1.1 DPPH radical-scavenging assay
Radical-scavenging ability of I. tinctoria plant extracts was measured by a method described previously[24] with slight modification. Briefly, the reaction mixture contains 2.5 mL of 0.004% DPPH solution and a range of diluted plant extracts (0.05–0.65 mg/mL) along with a tocopherol standard (1 mg/mL). Samples were incubation in the dark for 30 min before measuring the absorbance at 517 nm.
2.5.1.2 SO2-scavenging activity
SO2--scavenging activity was measured using a slight modification of the NBT reduction method[25]. In brief, the reaction mixture contained NBT (144 μmol/L), NADH (677 μmol/L), different dilutions of plant extracts (0.05–0.65 mg/mL) along with ascorbic acid and quercetin standards, and PMS (60 μmol/L). All reagents were prepared in 0.1 mol/L phosphate buffer, pH 7.4. All test tubes were incubated for 3 min and then the absorbance was measured spectrophotometrically at 560 nm.
2.5.1.3 NO2 radical-scavenging activity
The Griess-Ilosvay reaction method[26] was used, with minor modifications, to determine the NO2 radical-scavenging activity of plant extracts. The assay mixture (3 mL) consisted of sodium nitroprusside (SNP; 10 mmol/L), phosphate buffer (pH 7.4; 0.1 mol/L) and diluted sample extracts (0.05–0.65 mg/mL) along with rutin and ascorbic acid standards. Mixtures were incubated at 25 ℃ for 150 min. After incubation, 0.5 mL of the reaction mixture was mixed with 1 mL of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Next, 1 mL of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride was added and allowed to stand for 30 min. Then, the absorbance was measured spectrophotometrically at 564 nm.
2.5.1.4 OH–-scavenging activity
Hydroxyl radical-scavenging activity was measured following the protocol of Halliwell et al[27], in which hydroxyl radicals were generated via the fenton reaction, involving the Fe3+-H2O2 system. This assay depends on the degradation of 2-deoxyribose and condensation with TBA to give pink-colored complex. The reaction mixture contained EDTA (10 mmol/L), FeSO4·7H2O (10 mmol/L), 2-deoxyribose (10 mmol/L), diluted plant extracts (0.05–0.65 mg/mL) or mannitol standard, phosphate buffer (pH 7, 0.1 mol/L) and H2O2 (10 mmol/L), to make a final volume 1.8 mL. Reactions were incubated at 37 ℃ for 1 h. After this, 0.5% TBA (1 mL) and ice cold TCA (2.8% in 25 mmol/L NaOH) were added. After incubation at 80 ℃ for 30 min, the absorbance was measured spectrophotometrically at 532 nm.
2.5.2 Metal chelation
Metal-chelating activity of I. tinctoria extracts was measured by the ferrous ion (Fe2+) chelation method[28], in which Fe2+ in the reaction mixture was chelated by the antioxidants present in plant extracts. Chelation is induced by the addition of an Fe2+ source (ferrous sulfate). The remaining Fe2+ forms a red color complex with bipyridyl solution. The intensity of this complex depends on the chelating capacity of the plant extracts. In this assay, the reaction mixture contained diluted plant extracts (0.05–0.65 mg/mL) or EDTA standard, 0.5 mL FeSO4·7H2O (10 mmol/L), 1 mL Tris-HCl buffer, pH 7.4, 0.2 mol/L, 2-2′-bipyridyl solution (0.1% in 0.2 mol/L HCl), 0.4 mL hydroxylamine-HCl and 2 mL ethanol. The absorbance was measured spectrophotometrically at 522 nm.
2.6 Calculation of radical scavenging activity and half inhibitory concentration values
The percent scavenging and chelation activity were calculated using the following formula[29,30]:


where, Ac is the absorbance of the control (without plant extracts), and As is the absorbance of each plant extract dilution.
Half inhibitory concentration (IC50) of plant extracts for the above mentioned assays was calculated by plotting the concentration of plant extract against the corresponding % radical scavenging activity and fitting a linear model. From this equation for this model, the value of antioxidant to inhibit or scavenge 50% of free radical in a reaction mixture could be calculated.
2.7 In-vitro antioxidant assays
2.7.1 Ferric-reducing ability of plasm assay
Ferric-reducing ability of plasm (FRAP) assay was performed by using a modified method[31] that depends upon the change in absorbance at 593 nm as the colorless oxidized Fe3+ solution is reduced to the blue-colored Fe2+-TPTZ complex by antioxidants present in plant extracts. The stock solution included 300 mmol/L acetate buffer, pH 3.6, 10 mmol/L TPTZ in 40 mmol/L HCl and 20 mmol/L FeCl3·6H2O. The fresh FRAP reagent was prepared by mixing these reagents with 10:1:1 ratio. The temperature of working solution was raised to 37 ℃. Each diluted plant extract sample (0.05–0.65 mg/mL) and standard (FeSO4·7H2O) was allowed to react with FRAP reagent (2.85 mL) and incubated for 30 min in the dark. After incubation, spectrophotometric absorbance was measured at 593 nm. Results were expressed as μmol/L Fe2+/mg I. tinctoria extracts using the standard regression equation y = ax + b.
2.7.2 Total reducing power assay
Total reducing power of I. tinctoria plant extracts was determined[32] by reducing K3Fe(CN)6 via antioxidants present in plant samples. Increasing absorbance is tied to increases in the reducing power of the plant extracts. Briefly, each diluted plant extract sample (0.05–0.65 mg/mL) or rutin standard was mixed with 0.2 mol/L phosphate buffer, pH 6.6 and 1% potassium ferricyanide. Samples were incubated for 20 min at 50 ℃ before the addition of 2.5 mL 10% TCA. Next, 2.5 mL of the upper layer was collected, and diluted with 2.5 mL distilled water before the addition of 0.5 mL 0.1% FeCl3. Absorbance was measured spectrophotometrically at 700 nm.
2.7.3 Total antioxidant capacity
Total antioxidant capacity (TAC) of each plant extract was evaluated by the phosphomolybdenum method[33] with minor changes and compared to a gallic acid standard. In this assay, Mo(VI)-Mo(V) reduction via antioxidants that were present in samples of plant extract leads to the formation of a green phosphate/Mo(V) complex at an acidic pH; absorbance of this solution was measured spectrophotometrically at 695 nm. Briefly, diluted plant extracts (0.05–0.65 mg/mL) were combined with 1 mL of TAC solution. TAC solution was prepared by adding 0.6 mol/L sulfuric acid, 28 mmol/L NaH2PO4 and 4 mmol/L ammonium molybdate in 1:1:1 ratio. Then, all test tubes were incubated at 95 ℃ for 90 min. TAC is expressed as mg GAE/100 g I. tinctoria extracts and calculated via following formula:
A = cV/m
y = mx + c
where, A = amount of antioxidant compound (mg/g) in plant extract equivalent to gallic acid; c = concentration of gallic acid established from gallic acid calibration curve (mg/mL); x = absorbance and y = gallic acid equivalent; V = volume of sample (mL); m = weight of pure plant extracts (g).
2.8 Statistical analysis
All experimental outcomes are presented as mean ± standard deviation of three replicates. The data were analyzed using one-way analysis of variance (ANOVA); differences among samples were tested using Bonferroni’s multiple comparison tests using SPSS (version 16.0). All figures were plotted using Sigmaplot (version 8.0). Karl Pearson’s correlation analysis (r2) for correlation was performed between antioxidant activity and quantitative measurements by using SPSS. P < 0.05 was considered significant.

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3 Results

3.1 Quantitative analysis of phytoconstituents
Samples of I. tinctoria from both extracts had appreciable amounts of phenolic compounds, but hydroethanolic had slightly higher content ((212.920 ± 0.002) mg/g) in comparison to the ethanol extract ((211.690 ± 0.004) mg/g) and differences were statistically significant when compared with the standard group (P < 0.001, post-hoc analysis; Bonferroni multiple comparision test, P = 0.000, df = 2, 6). This difference may be due to the mixture of 2 different solvents having different polarities, or may be due to differences in the solubility of these compounds in organic and aqueous solvents. TPC was determined by using standard calibration curve (Figure 1A), having a regression equation of y = 0.158 x + 0.071 and a correlation coefficient (r2) 0.979.
Total flavonoid content of I. tinctoria extracts was greater in HE extract ((149.770 ± 0.002) mg/g) than in ethanol extract ((132.603 ± 0.002) mg/g) and differences were significant with respect of standard, rutin (P < 0.001, post-hoc analysis; Bonferroni test, P = 0.000, df = 2, 6). TFC was determined by using standard (rutin) calibration curve, which was fit with a linear regression (y = 0.775 x + 0.060; r2 = 0.950; Figure 1B).
TSC was determined by using a plant-saponin standard calibration curve (y = 0.062 x + 0.033, r2 = 0.954). Saponin content in hydroethanol was (100.911±0.010) mg/g whereas ethanol extract had (159.680±0.005) mg/g of plant extract (Figure 1C). Differences were significant compared with standard group, i.e., plant saponin (P < 0.001, post-hoc analysis; Bonferroni test, P = 0.000, df = 2, 6).
TTC and proanthocyanidine (condensed tannins) content in I. tinctoria extracts were determined from standard calibration curves (Figure 1D and Figure 1E) of tannic acid and rutin respectively (regression equations: tannic acid y = 0.049 x + 0.093, r2 = 0.935; rutin y = 0.115 x + 0.007, r2 = 0.915). TTC in hydroethanol and ethanol extracts was (229.112 ± 0.000) and (230.440 ± 0.000) mg/g (P < 0.001 vs standard group, tannic acid, post-hoc analysis; Bonferroni multiple comparison test, P = 0.000, df = 2, 6), whereas proanthocyanidine content was (2.589 ± 0.071) and (13.380 ± 0.001) mg/g respectively (differences were significant for ethanol extract, P < 0.001 vs standard group rutin but insignificant for hydroethanol extract, post-hoc analysis; Bonferroni multiple comparison test, P = 0.000 for ethanol whereas 0.725 for hydroethanol, df = 2, 6).
3.2 Antioxidant prospective
3.2.1 DPPH-scavenging activity
I. tinctoria extracts showed considerable scavenging capacity (Figure 2). A positive linear relationship between DPPH-scavenging ability of plant extracts and quantitative measurements was dependent on phytoconstituents (Table 1). The highest correlation was 0.988 between flavonoid content and DPPH, which clearly indicated that the scavenging property of plant extracts may be due to the presence of bioactive compounds. IC50 values of hydroethanol and ethanol extracts were 829 and 1 306.1 μg/mL, which were both significantly higher than those of the standard, tocopherol (90 μg/mL).
3.2.2 Superoxide anion-scavenging activity
I. tinctoria extracts showed good scavenging potential of superoxide anions when compared with standards, ascorbic acid and quercetin (Figure 3). Percentage scavenging activity increases with rise in concentration. IC50 values for hydroethanolic and ethanol crude extracts were 638.1 and 628.1 μg/mL respectively whereas those for both standards (i.e., ascorbic acid and quercetin) were 194.3 and 463.8 μg/mL. Differences were highly significant compared with the standard groups.
3.2.3 NO2-scavenging activity
Figure 4 shows the NO2-scavenging activity of I. tinctoria plant extracts. Ethanol extracts had higher scavenging activity than both hydroethanol extract and the ascorbic acid standard, but lower activity than rutin (P<0.001). IC50 values for hydroethanolic and ethanolic were 978 and 43.31 μg/mL respectively, showing antioxidant activity, which was quite near to the antioxidant activity of rutin (28.4 μg/mL).
3.2.4 Hydroxyl ion-scavenging activity
Both extracts of I. tinctoria possessed high hydroxyl-scavenging potential, relative to the mannitol standard (Figure 5). IC50 values for hydroethanolic and ethanolic extracts were 26.7 and 29.4 μg/mL, which were significantly lower than mannitol (1 044 μg/mL), suggesting that they might be effective at reducing hydroxyl radical-related oxidative cell damage.
3.2.5 Metal-chelating activity
The metal chelating capacities of I. tinctoria ethanol and hydroethanol extracts increased with increasing extract concentration. Hydroethanol extracts had high chelating activity in comparison with the ethanol extracts (Figure 6).
3.2.6 Total antioxidant activity (FRAP)
A regression equation for FeSO4 was calculated (y = 3.166 x – 0.193), and used to evaluate FRAP values of ethanolic and hydroethanolic. Hydroethanolic extract had significantly (P < 0.001 vs standard group; P < 0.05 vs ethanol extract), higher ((17.064 ± 0.021) μmol/L Fe2+/mg Indigofera tinctoria extracts) ferric ion (Fe3+) reducing capability than ethanol extract ((16.170 ± 0.005) μmol/L Fe2+/mg) (P < 0.01 vs standard group), indicating its potent antioxidant activity. Correlation analysis with other quantitative parameters also showed positive linear relationships among parameters. The highest correlations were 0.994 (99.4%) for phenolic content of ethanolic extract and flavonoid content in the hydroethanolic extract.
3.2.7 Reducing power assay
Results show lower reducing activity in I. tinctoria plant extracts in comparison to the rutin standard. The hydroethanol extract had slightly higher reducing activity in comparison to the ethanol extract. An increase in absorbance was observed with increase in extract concentration (mg/mL). Both plant extracts were able to reduce Fe3+ ions, but at a lower rate than the standard (Figure 7). Both hydroethanol and ethanol extracts showed significant increment in their reducing activity (P < 0.01 vs rutin post-hoc analysis, Bonferroni multiple comparision test).
3.2.8 Total antioxidant capacity
Hydroethanol extract of I. tinctoria showed significantly greater (P < 0.01) TAC values ((802.670 ± 0.005) mg GAE/g of plant extract) in comparison to ethanol extracts (P < 0.05, (575.370 ± 0.003) mg GAE/g of plant extract).

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4 Discussion

Plant polyphenols, a group of important secondary metabolites, possess potent antioxidant properties that participate in defense mechanisms of the plant. Studies have shown that DNA damage caused by oxidative stress can be reduced by the consumption of phenol-rich diets[34]. Polyphenols are good electron donors that have the ability to terminate the free radical chain reaction by converting them into more stable compounds. Saponins, important glycosides, containing either terpenoid or steroidal aglycone moieties and sugar chains, possess several therapeutic activities like permeabilization of cell membrane[35], lowering of cholesterol, stimulation of luteinizing hormone (LH) release leading to abortifacient properties[36], immunomodulatory activity[37], antitumor property[38] and adjuvant property[39]. Various phytoconstituents, due to several biological functions, find an important place in the defense category. Quantitative result of phytoconstituents in hydroethanolic and ethanolic extracts of I. tinctoria showed appreciable amount of polyphenols (phenols and flavonoids), saponins, tannins and condensed tannins (proanthocyanidines) and good antioxidant efficacy, making this plant valuable in the world of herbal medicine.
During several metabolic processes, various reactive species of O2- and their derivatives are generated as a byproduct. Some contain unpaired valence shell electrons like nitric oxide, nitrogen dioxide, peroxynitrite, hydroxyl, superoxide and peroxyl radicals, whereas some are not free radicals, but act as a reservoir to produce other highly reactive oxygen products. These oxygen derivatives are highly reactive and depend on cascades of redox systems, mitochondrial electron transport chain as well as various metabolic reactions like lipid peroxidation[40]. ROS concentration is controlled by various ROS-scavenging pathways in aerobic organisms, but its imbalance may give rise to cell dysfunction, DNA damage, metabolic obliterations and cell death[41] that are collectively known as “oxidative stress.”
Superoxide, hydroxyl and nitric-oxide radicals are highly toxic ROS that can cause various health-related problems, so it is important to evaluate the scavenging properties of herbal medicines against these important free radicals. Hydroethanolic and ethanol extracts of I. tinctoria showed remarkable radical scavenging and antioxidant activity against various ROS.
DPPH, a stable free radical, possesses the property of delocalization and provides a deep violet color and undimerization capacity that makes it stable at ambient temperature. Antioxidants that possess electron-donating capacity reduce this radical, forming a yellow color. Various free radicals like superoxide, nitric oxide, hydroxyl radicals are toxic ROS that have capability to disrupt various signaling pathways and can lead to cell death. The bivalent metal ion transition specially ferrous ion initiates lipid peroxidation and decomposition of lipid peroxides into peroxyl and alkoxyl radicals that further add to oxidative stress[42]. Both extracts (hydroethanolic and ethanolic) of I. tinctoria possess good scavenging activity against these free radicals. This denotes its potential to reduce cell damage.
Today, in order to combat severe organ-related disorders, metabolic dysfunction and formation and accumulation of undesired harmful compounds, the use of antioxidant supplements may provide an important resource[43]. Total antioxidant capacity of I. tinctoria extracts was determined by the formation of a green phosphate/Mo (V) complex. It was noticed that both extracts of I. tinctoria exhibited good antioxidant capacity in comparison to the gallic acid standard.
Reducing power of a plant extract is an important property that correlates with its antioxidant activity in a concentration-dependent manner. The reducing property of antioxidant depends upon its electron-donating capacity and is ultimately converted into more stable form thus helpful in terminating the free radical chain reaction. Both extracts of I. tinctoria had reducing power that increased proportionally with concentration; absorbance increased with increasing concentration of extract and also the FRAP.
A comparative study of the presence of phenolics, tannins and flavanoids in different extracts of I. tinctoria (petroleum ether, benzene, chloroform, ethyl acetate, methanol and water), along with our study, clearly indicates that ethanolic and hydroethanolic extracts had the greatest tannin and flavonoid contents relative to all above mentioned extracts. The percentage yield of hydroethanolic extract was also found to be very high (25.8%) in comparison to other mentioned extracts[17].
Correlation analysis between bioactive constituents (phenols, flavonoids, saponins, and tannins) and scavenging assays (DPPH and FRAP) also revealed good positive correlations that suggest the possible dependency of antioxidant activity on the presence of bioactive compounds. The highest correlations of HE extract are 0.988 and 0.994 for DPPH and FRAP assays that denote the possible major role of flavanoids in scavenging assays. In the case of ethanol extract, the highest correlations (0.970 and 0.994 for DPPH and FRAP, respectively) indicated the possible major involvement of phenols in scavenging assays. Other remaining bioactive compounds were also positively correlated to the antioxidant and scavenging potential of I. tinctoria hydroethanolic and ethanolic extracts. IC50 values of hydroethanolic extract in comparison of ethanolic extract for various scavenging assays also indicate that this extract presents an option for future studies to evaluate its use in the treatment of several health problems.
Previous studies reported that I. tinctoria had good antibacterial[44] and antihepatotoxic activities[45]. Six different rotenoids from I. tinctoria were characterized[46] and their efficacy against larvae of Anopheles stephensi and adults of Callosobruchus chinensis was explored. Two important antibacterial compounds from the methanol extract[47] were also isolated and named “indicant” and “sumatrol”. Indigotine, another fractionated compound from I. tinctoria petroleum ether extract, also showed hepatoprotective activity[48]. All these reports point out strong medicinal potential of I. tinctoria. Briefly, results showed that both ethanol and hydroethanolic extracts of I. tinctoria had high levels of bioactive compounds, and good antioxidant and scavenging potential. Our study revealed that I. tinctoria contains various bioactive constituents, some of which may be useful in combating various severe health problems. Some compounds have already been isolated from various extracts but more remains to be discovered.

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-  References
  

5 Conclusion

In conclusion, this study expanded the current knowledge of bioactive constituents, antioxidant potential and free radical-scavenging activity of both ethanolic and hydroethanolic extracts of I. tinctoria. This indicates the positive way to researcher to isolate and characterize various bioactive compounds that may have potent antioxidant activity. Further research on this plant may be helpful in treating various health problems.

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6 Acknowledgements

We are very thankful to technical and laboratory staff of Department of Bioscience and Biotechnology to give support to complete the study.

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7 Competing interests

Authors have no conflict of interests.

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Figures and Tables in this article:



Figure 1 Calibration curve of phenolic (A), flavonoid (B), saponin (C), tannin (D) and proanthocyanidine (E) contents


 Figure 2 DPPH-scavenging activity of I. tinctoria plant extracts

DPPH: 2,2-diphenyl-1-picrylhydrazyl.
 

Figure 3 Superoxide radical-scavenging activity of I. tinctoria plant extracts


Figure 4 NO2-scavenging activity of I. tinctoria plant extracts


Figure 5 Hydroxyl radical-scavenging activity I. tinctoria plant extracts


Figure 6 Metal-chelating activity I. tinctoria plant extracts


Figure 7 Reducing power assay I. tinctoria plant extracts
 

Table 1 Correlation analysis (r2) between phytoconstituents and antioxidant parameters of I. tinctoria extracts

DPPH: 2,2-diphenyl-1-picrylhydrazyl; FRAP: ferric-reducing ability of plasma.

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