*Corresponding Author:
S. R. Pai
Regional Medical Research Centre, ICMR, Nehru Nagar
E-mail: [email protected]
Date of Submission 29 January 2013
Date of Revision 23 May 2013
Date of Acceptance 25 May 2013
Indian J Pharm Sci 2013;75(4): 483-486  

Abstract

Aim of the study was to evaluate antioxidant activity and total phenolic content of Achyranthes coynei; an endemic plant used in treatment of several diseases in the same lines that of Achyranthes aspera by traditional practitioners of Belgaum region. Efficiency of extraction methods was studied for aerial parts (leaves, stem, and inflorescence) extracted in methanol using continuous shaking, microwave assisted and ultra sonic extraction technique, by exposing it for different time period. Total phenolic content was measured by Folin-Ciocalteu method and antioxidant activity using 2,2'-diphenyl-1-picryl hydrazyl radical scavenging assay and ferric reducing antioxidant power assay. Extracts of A. coynei revealed highest yield of total phenolic content in continuous shaking method compared to other methods. Significantly higher amount of phenolic content (467.07Î23.35 tannic acid equivalent and 360.83Î18.04 caffic acid equivalent mg/100 g FW) was estimated at 360 min of continuous shaking extraction. In 2,2'-diphenyl-1-picryl hydrazyl radical scavenging assay and ferric reducing antioxidant power assay, inflorescence and leaf showed highest potential activity, respectively. Stem extracts showed lower yield of total phenolic content and antioxidant activity. Results also showed 2,2'-diphenyl-1-picryl hydrazyl radical scavenging assay had significant correlation with total phenolic content. This is first report of total phenolic content and antioxidant studies in A. coynei.

Keywords

Antioxidant activity, Achyranthes coynei, endemic, total phenolic content

Plants are good source of biologically active secondary metabolites which have many therapeutic potential in many diseases and even in free radical associated disorders [1]. Among secondary metabolites synthesized, plant polyphenols are the aromatic hydroxylated compounds which have the most potent and therapeutically useful bioactive substances. Promising radical scavenging ability of the phenolic compounds produced in higher plants is studied extensively [2,3]. Oxidation stress is one of the major concerns of health in modern era and antioxidants have been reported to prevent oxidative damage caused by free radical, via interfering with the oxidation process by reacting with free radicals, by chelating with catalytic metals, and also by acting as oxygen scavengers [4,5]. Although several synthetic antioxidants, such as butylated hydroxyanisole and butylated hydroxytoluene, are available because of their toxicity problems; there is an upsurge of interest in the therapeutic potentials of plants as antioxidants.

In addition to the natural antioxidants like vegetables, fruits, spices and tea, scientific evaluation of plant’s properties through potent pharmacological activities, toxicity profiling and economic viability are needed for growing recognition for medicinal plants and herbal products as novel antioxidants in recent decades. Therefore, significant consideration has been directed toward the detection of antioxidant properties in plant species.

Achyranthes coynei Sant. is a rare, endemic plant species belonging to family Amaranthaceae. Its distribution was restricted to Maharashtra and recently was reported from Karnataka [6]. Achyranthes aspera L. is the much known medicinal plant from the family used in treating various disorders [7]. Achyranthes coynei, locally known as “Kempu uttarani” is used as substitute for A. aspera by local traditional practitioners in similar disease treatment because of comparable appearance [6].

Green leaves, stem, and inflorescences of A. coynei were obtained from a single produce at Pachapur, from Belgaum and a specimen was authenticated and deposited at Herbaria, Regional Medical Research Centre, Belgaum (Voucher Number: RMRC‑785). Three extraction methods with three exposure times were compared for their antioxidant activity and the yield of phenolics. For all methods 1 g of plant materials (green leaves, stem, and inflorescence) were extracted with 20 ml of 95% methanol. Continuous shaking extraction (CSE) was carried out on orbital shaker (Remi Instruments, Mumbai, India) at a constant stirring of 110 rpm at room temperature for 30, 180, and 360 min separately. Microwave assisted extraction (MAE) was carried out at 1, 3, and 5 min of exposure using microwave oven (Godrej, GM×30 CA1 SIM) at 180 W. The suspensions were cooled after every 1 min to avoid bumping of solvent out of the flask in order to complete 3 and 5 min of microwave exposure. Ultrasonic extraction (USE) was performed on ultrasonic bath (Soncis Vibracell, USA) 130 Watt, at working amplitude of 60 Khz. The samples were exposed for 5, 15, and 30 min of sonication at room temperature. These extracts were filtered using Watman filter paper No. 1 and volume was made up to 20 ml with 95% methanol.

Total phenolic content (TPC) was quantified using modified Folin–Ciocalteu method described by Wolfe et al. [8]. The absorbance of blue color was read at 760 nm on double beam spectrophotometer. The results were compared to the standard curve and were expressed as mg tannic and/or caffeic acid equivalent per 100 g fresh plant material.

Antioxidant activities were determined as the measure of free radical scavenging activity using 2,2'‑diphenyl‑1‑picryl hydrazyl (DPPH) assay as determined by Brand‑Williams et al. [9]. The absorbance at 515 nm was measured using methanol as blank and DDPH radical scavenging activity was calculated. Ferric reducing antioxidant power (FRAP) assay was used to measure the total antioxidant power. Antioxidant assay was preformed as previously described and absorbance was taken at 593 nm [10]. The results were expressed as ascorbic acid equivalent antioxidant capacity (AEAC) and Trolox equivalent antioxidant capacity (TEAC) as determined by Gil et al. [11].

The values are represented as mean±SD of three individual readings. The calibration curves for all standards were generated with the correlation coefficients (R2) above 0.9500. The regression equations for various determinations are given as follows, for TPC: tannic acid, y=0.003x+0.115; caffeic acid y=0.004x+0.092; for DPPH assay: TEAC, y=0.000x+0.001; AEAC, y=0.000x-0.024; for FRAP assay: TEAC, y=0.000x+0.152; AEAC, y=0.000x+0.114. The respective contents were calculated using above equations from standard calibration curves.

Estimated TPC from various parts of A. coynei extracted using CSE, MAE, and USE methods are depicted in Table 1. Total phenolic content ranged from 85.13±4.26 to 467.07±23.35 mg tannic acid equivalent (TAE)/100 g and 65.77±03.29 to 360.83±18.04 mg caffeic acid equivalent (CAE)/100 g on fresh weight basis. TPC in stem was lowest from 85.13±4.26 mg TAE/100 g and 65.77±3.29 mg CAE/100 g at 30 min to highest of 190.02±9.50 mg TAE/100 g and 146.80±7.34 mg CAE/100 g at 360 min of CSE. TPC in leaves ranged from 220.79±11.04 to 354.51±17.73 mg TAE/100g and 170.57±8.53 to 273.88±13.69 mg CAE/100g. Among all parts and extraction methods tested highest TPC was observed in inflorescence (467.07±23.35 TAE and 360.83±18.04 CAE mg/100 g) at 360 min of CSE method and lowest was recorded in stem. Synchronized patterns of increase in TPC with respect to time of extraction were observed for inflorescence in CSE and USE; stem in CSE and MAE and leaf in MAE and USE methods.

  Method of
extraction
Plant parts Time
min  
Total phenolic content
mg/100g FW
Tannic
acid ± sd  
Caffeicacid ± sd
Continuous
shaking
extraction
      Leaf   30 312.67 ± 15.63 241.56 ± 12.08
180 354.51 ± 17.73 273.88 ± 13.69
360 315.59 ± 15.78 243.81 ± 12.19
          Stem   30 086.10 ± 04.31 066.52 ± 03.33
180 142.35 ± 07.12 109.98 ± 05.50
360 190.02 ± 09.50 146.80 ± 07.34
    Inflorescence   30 302.90 ± 15.15 234.01 ± 11.70
180 368.44 ± 18.42 284.64 ± 14.23
360 467.07 ± 23.35 360.83 ± 18.04
Microwave
assisted
extraction
Leaf   1 295.94 ± 14.80 228.63 ± 11.43
3 270.08 ± 13.50 208.65 ± 10.43
5 324.39 ± 16.22 250.61 ± 12.53
      Stem   1 115.90 ± 05.80 089.54 ± 04.48
3 130.15 ± 06.51 100.55 ± 05.03
5 145.43 ± 07.27 112.35 ± 05.62
Inflorescence   1 250.32 ± 12.52 193.39 ± 09.67
3 411.14 ± 20.56 317.63 ± 15.88
5 249.67 ± 12.48 192.89 ± 09.64
Ultra sonic
extraction
Leaf   5 220.79 ± 11.04 170.57 ± 08.53
15 290.65 ± 14.53 224.54 ± 11.23
        30 300.42 ± 15.02 232.09 ± 11.60
Stem   5 085.13 ± 04.26 065.77 ± 03.29
15 107.27 ± 05.36 082.87 ± 04.14
30 101.38 ± 05.07 078.32 ± 03.92
Inflorescence   5 297.23 ± 14.86 229.63 ± 11.48
15 303.17 ± 15.16 234.22 ± 11.71
30 364.98 ± 18.25 281.97 ± 14.10

Table 1: Efficiency of extraction methods on Content of total phenolics in various parts of A. Coynei

Antioxidant potential of aerial parts of A. coynei using different extraction methods were tested using DPPH and FRAP assay and results were presented in Table 2. Ascorbic acid equivalent activity was recorded higher over trolox in both antioxidant assays for the tested extracts. The DDPH radical scavenging activity was highest in inflorescence extract with CES 360 min (TEAC 473.63 μM and AEAC 666.43 μM) as per Table 2. The results were in correlation to the phenolic content estimated. Increase in DPPH activity with respect to time (30-360 min) was observed in CSE and MAE methods. Minor fluctuation in the activity was observed for extracts exposed to USE method. However, it was interesting to note that in CSE yielded higher and significant results over the other methods tested.

Method of extraction Plant parts Time min DPPH µM FRAP µM
TEAC ± SD              AEAC ± SD TEAC ± SD              AEAC ± SD
Continuous shaking Leaf 330 239.17 ± 11.96 337.04 ± 16.85 462.14 ± 23.11 566.26 ± 28.31
extraction   180 386.44 ± 19.32 544.57 ± 27.23 688.82 ± 34.44 844.02 ± 42.20
    360 432.39 ± 21.62 609.32 ± 30.47 508.37 ± 25.42 622.91 ± 31.15
  Stem 30 110.16 ± 05.51 155.24 ± 07.76 173.98 ± 08.70 213.18 ± 10.66
    180 208.54 ± 10.43 293.87 ± 14.69 286.28 ± 14.31 350.78 ± 17.54
    360 291.6 ± 14.58 410.92 ± 20.55 293.59 ± 14.68 359.73 ± 17.99
  Inflorescence 30 231.51 ± 11.58 326.25 ± 16.31 254.55 ± 12.73 311.90 ± 15.60
    180 418.84 ± 20.94 590.23 ± 29.51 451.70 ± 22.59 553.47 ± 27.67
    360 473.63 ± 23.68 667.43 ± 33.37 547.82 ± 27.39 671.25 ± 33.56
Microwave assisted Leaf 1 315.75 ± 15.79 444.96 ± 22.25 491.05 ± 24.55 601.69 ± 30.08
extraction   3 304.56 ± 15.23 429.18 ± 21.46 515.89 ± 25.79 632.12 ± 31.61
    5 374.07 ± 18.70 527.14 ± 26.36 586.44 ± 29.32 718.57 ± 35.93
  Stem 1 169.66 ± 08.48 239.08 ± 11.95 193.81 ± 09.69 237.48 ± 11.87
    3 195.58 ± 09.78 275.61 ± 13.78 239.11 ± 11.96 292.98 ± 14.65
    5 202.06 ± 10.10 284.74 ± 14.24 231.80 ± 11.59 284.03 ± 14.20
  Inflorescence 1 220.91 ± 11.05 311.30 ± 15.57 251.00 ± 12.55 307.56 ± 15.38
    3 353.45 ± 17.67 498.08 ± 24.90 350.26 ± 17.51 429.17 ± 21.46
    5 196.17 ± 09.81 276.44 ± 13.82 228.36 ± 11.42 279.81 ± 13.99
Ultra sonic extraction Leaf 5 229.74 ± 11.49 323.75 ± 16.19 391.69 ± 19.58 479.94 ± 24.00
    15 284.53 ± 14.23 400.96 ± 20.05 499.61 ± 24.98 612.17 ± 30.61
    30 311.04 ± 15.55 438.31 ± 21.92 493.66 ± 24.68 604.88 ± 30.24
  Stem 5 107.80 ± 05.39 151.92 ± 07.60 286.28 ± 14.31 350.78 ± 17.54
    15 139.61 ± 06.98 196.74 ± 09.84 254.24 ± 12.71 311.52 ± 15.58
    30 132.54 ± 06.63 186.78 ± 09.34 204.87 ± 10.24 251.03 ± 12.55
  Inflorescence 5 237.40 ± 11.87 334.55 ± 16.73 266.55 ± 13.33 326.61 ± 16.33
    15 259.20 ± 12.96 365.26 ± 18.26 295.46 ± 14.77 362.03 ± 18.10
    30 295.13 ± 14.76 415.90 ± 20.80 327.09 ± 16.35 400.78 ± 20.04

Table 2: Efficiency of Extraction methods on dpph radical scavenging and frap antioxidant Activities in various parts of A. Coynei

Ferric reducing capacity was higher in leaf sample extracted using 180 min of CSE method (688.82±34.44 μM TEAC and 844.02±42.20 μM AEAC). The pattern observed for the FRAP activity was unlike that of DPPH and TPC. Increase in activity was proportional to the immediate next exposure time in series for all the extraction methods followed by drop or no change (Table 2). This may be because extended exposure time in any extraction method affecting activity. Small variation in activity was noticed with change in exposure times for a particular method and plant part. Leaf yielded highest activity for FRAP over stem and inflorescence.

Different levels reported in this study may be attributed to the different plant parts and extraction methods with time used to express as total phenolic contents. In general, there was a significant correlation between TPC and DPPH scavenging assays over FRAP. Findings of the study also indicate polyphenols are important contributors in free radical scavenging activities. The results are in accordance with those carried out in other plants [12].

Hagerman et al. [13] reported that the high molecular weight phenolics (tannins) have potent scavenging activity toward the free radicals and that the activity depends on the molecular weight, the number of aromatic rings, and nature of hydroxyl groups. Therefore, antioxidant activities of these extracts cannot be predicted on the basis of their TPC alone, but will also require proper characterization of individual phenolic components.

The present study reports TPC and antioxidant activity of A. coynei for the first time. Results showed antioxidant potential of aerial parts using different extraction methods. The study may support use of A. coynei to prevent in vivo oxidative damage associated with illnesses. Present study also suggests further need for detailed phytochemical investigation and pharmacological studies to support use of this plant by traditional practitioners.

Acknowledgements

Authors are grateful to the Director‑in‑Charge, Regional Medical Research Centre (ICMR) Belgaum, for providing facilities. VU and SRP are grateful to the Indian Council of Medical Research, for SRF and PDF financial grants, respectively. Authors extend their thanks to Dr. Subarna Roy and Dr. Rajesh Joshi RMRC, Belgaum for their support and suggestions.

References