Basic, Applied Pharmacy and Pharmacology

RP-HPLC and GC-MS Based Identification of Phenolic Acids, Flavonoids and Hydroxyl Containing Compounds from One of the Lead Accessions of Amaranthus hypochondriacus L. Identified on the Basis of Biomarkers of Antioxidant Potential

Soumen Bhattacharjee*, Manashi Aditya1, Tapan Sil2

*Plant Physiology & Biochemistry Research laboratory, UGC Centre for Advanced Study, Department of Botany, The University of Burdwan, Burdwan, West Bengal, India

1Present address: Post Graduate Department of Botany, Hooghly Mohsin College (WBES), Chinsurah, Hooghly, West Bengal, India

2Plant Chemistry Department, Botanical Survey of India, A. J. C. Bose Indian Botanic Garden, Shibpur, Howrah, India

Corresponding authors

Soumen Bhattacharjee
Plant Physiology & Biochemistry Research Laboratory
UGC Centre for Advanced Study
Department of Botany
The University of Burdwan
Burdwan, West Bengal, India
Tel: 9433778493
E-mail: soumen1995@yahoo.com/
sbhattacharjee@bot.buruniv.ac.in

  • Received Date: April 25, 2018
  • Accepted Date: May 22, 2018
  • Published Date: May 26, 2018

DOI:   10.31021/bapp.20181102

Article Type:  Research Article

Manuscript ID:   BAPP-1-102

Publisher:   Boffin Access Limited

Volume:   1.1

Journal Type:   Open Access

Copyright:   © 2018 Aditya M, et al.
Creative Commons Attribution 4.0


Citation

Aditya M, Sil T, Bhattacharjee S, (2018) RP-HPLC and GC-MS based Identification of Phenolic Acids, Flavonoids and Hydroxyl Containing Compounds from One of the Lead Accessions of Amaranthus hypochondriacus L. Identified on the Basis of Biomarkers of Antioxidant Potential. Basic Appl Pharm Pharmacol. 2018 May;1(1):102.

Abstract

A comparative evaluation of anti-lipid peroxidation property, reducing power, metal chelating activity, hydroxyl radical scavenging property and total antioxidant capacity of leaf extracts of ten experimental accessions of Amaranthus hypochondriacus L. (EC42352, IC47434, IC94661, IC95251, IC95316, IC95322, IC95326, IC107144, EC146543 and IC42397) revealed significantly higher antioxidant availability in the red accession IC107144. Selective Reverse Phase-High Performance Liquid Chromatography (RPHPLC) for some important health promoting phenolic acids and flavonoids of the lead accession demonstrated the significant availability of gallic acid, caffeic acid, syringic acid, p-coumaric acid, ferullic acid, rutin, kaempferol and elagic acid. Gas Chromatography-Mass Spectroscopy (GC-MS) study exhibited presence of several phytochemicals containing hydroxyls, responsible for radical scavenging properties.

Keywords

Amaranth; Antioxidant capacity; Anti-lipid peroxidation property; Reducing power; Metal chelating property; GC-MS and HPLC analysis

Introduction

Plant natural products such as phenolic acids, flavonoids have received much attention as therapeutic agent to fight against degenerative diseases mediated by oxidative stress [1-3]. Phenolic compounds, in general execute their therapeutic activities primarily because of their potential antioxidative and anti-inflammatory properties [1,2,4]. Loss of redox homeostasis which is an obvious manifestation of any kind of degenerative diseases caused oxidative deterioration of infected cells and tissues [5,6]. Antioxidant therapy offers an effective path to eliminate the endogenous titer of Reactive Oxygen Species (ROS) for restoring redox homeostasis and minimizing oxidative damage [6-9]. Antioxidants are substances which when supplemented in low concentrations restore redox homeostasis and significantly delay or prevent oxidative deterioration [6,10]. Antioxidant apart from their bona-fide ROS scavenging properties can prevent or delay oxidation of food stuff which are normally initiated by exposure of food to unfavourable environmental factors such as light temperature and air [11]. Moreover, restriction of commonly used synthetic antioxidants like Butylated Hydroxyl Tolune (BHT), Butylated Hydroxyl Anisol (BHA), Tert-Butyl Hydroquinone (TBHQ) justified the hunch to find out the natural source of antioxidants.

Among the naturally occurring antioxidants, phenolics, flavonoids and various antioxidative pigments such as carotenoids anthocyanins, betacyanins are most important. Polyphenols particularly the flavonoids have the ability to scavenge ROS and also to chelate the transition metal ions necessary for the generation of OH· through Fenton reaction [12]. Phenolics typically exhibit chain reaction breaking properties. Infact all these polyphenolic compounds flavonoids and the antioxidative pigments exhibit anti-lipid peroxidation properties either by inactivating lipoxigenase or through scavenging ROS necessary for propagation reactions associated with lipid peroxidation [12,13]. All these antioxidants also exhibit significant reducing properties through donation of electrons to stabilize the ROS and breaking the free radical mediated chain reactions [14].

The nutritive value of the pseudocereal amaranth and their potential use as functional food is well recognized and gaining momentum [15-18]. Availability of antioxidant like phenolic acid, flavonoids and other polyphenolic compounds along with good quality of proteins, fatty acids, and fibres have been reported from some important leaf and grain amaranth. Several worker [15,16,18-21], in their studies reported presence of several classes of phenolic acids and flavonoids from the seeds of Amaranthus hypochondriacus and Amaranthus caudatus. Further the antioxidant potential of seed extract was confirmed by their radical scavenging property [22,23] but no detail analysis of foliar antioxidative potential were assessed in terms of availability of antioxidant like phenolic acids and flavonoids, their reducing property, metal chelating property and anti-lipid peroxidation properties.

An efficient use of plant genetic resource for crop improvement based on availability and nutrition require a systematic exploration of nutritional attributes in diverse land races and cultivars. In spite of huge potential to address the problem of food security as functional food, the genetic diversity-based analysis of nutritional attributes particularly the antioxidative potential of amaranth are yet to be exploited.

Therefore, the present investigation makes an effort to assess and compare the antioxidant potential in terms of important antioxidant properties like anti-lipid peroxidation, reducing property, metal chelating and radical scavenging properties of ten promising accessions of A. hypochondriacus. Further, the present study also aims at Phase-High Performance Liquid Chromatography (RP-HPLC) based identification and quantification of some bona-fide health promoting phenolic acids and flavonoid from the lead accession identified on the basis of above mentioned criteria. GC-MS study was also conducted to asses simultaneously hydroxyl containing compounds with antioxidant properties from the lead accession.

Materials and Methods

Seeds of ten different accessions of A. hypochondriacus L. (EC42352, IC47434, IC94661, IC95251, IC95316, IC95322, IC95326, IC107144, EC146543 and IC42397) were collected from National Bureau of Plant Genome Research (NBPGR), New Delhi, India and were cultivated in Crop Research and seed Multiplication Farm, University of Burdwan, West Bengal, India, based on Standard Procedure.

Sample preparation for Gas Chromatography-Mass Spectroscopy (GC-MS) study

20 grams of oven dried and powdered leaves of each experimental material were extracted with 200 ml 95% ethanol for 12 hours and filtered through Whatman filter paper #1 (41 cm) with 2 gram sodium sulphate (before filtering, the filter paper was wetted with 95% ethanol containing sodium sulphate). The filtrate collected was concentrated by bubbling N2 gas into the solution in a rotary vacuum evaporator. 2 μl of solution was taken for GC-MS study.

GC-MS Condition

Qualitative antioxidant profiling for hydroxyl containing flavonoids were done as per the procedure of Aditya & Bhattacharjee (2018) [24], with Shimadzu GC-MS QP2010 system comprising a gas chromatograph interfused by a MS employing the following conditions: Fused silica column (30 × 0.25 mm) 1D × 1EMdf with 100% Dimethyl polysiloxane, operating in electron impact mode at 70ex, He (99.99%) as carrier gas at constant flow of 1 ml/min and injection volume of 0.5EI (split ratio 1:1); injector temperature 250°C, ion source temperature 280°C. Oven temperature was programmed from 110°C (isothermal for 2 min) with an increase of 10°C/min, to 200°C, then 5°C to 280°C, ending with 9 min isotherm at 280°C.

Sample preparation for HPLC study

15 grams of oven dried and powdered leaves of each experimental material were extracted with 150 ml 95% ethanol for three to four cycles by using soxhlet apparatus. The extract was collected and filtered. The filtrate was concentrated and dried at 50 ± 2°C under rotary vacuum evaporator (Eyela). The dried crude extract was dissolved in mobile phase. After filtering through filter paper (membrane filter, Millipore), the extract was injected into HPLC.

RP -HPLC analysis of phenolic acids and flavonoids

HPLC analyses were performed using Dionex Ultimate 3000 liquid chromatography including a Diode Array Detector (DAD) with 5 cm flow cell and with Chromeleon system manager as data processor. Separation was achieved by a reversed-phase Acclaim C18 column (5 μ particle size, 250 × 4.6 mm). 20 μL of sample was introduced into the HPLC column.

The mobile phase contains methanol (Solvent A) and 0.5% aqueous acetic acid solution (Solvent B) and the column was thermostatically controlled at 25°C and the injection volume was kept at 20 μl. A gradient elution was performed by varying the proportion of solvent A to solvent B. The gradient elusion was 10% A and 90% B with flow rate 1 ml/min to 0.7 ml/min in 27 min, from 10 to 40% A with flow rate 0.7 ml/min for 23 min, 40% A and 60% B with flow rate 0.7 ml/min initially for 2 min and then flow rate changed from 0.7 to 0.3 ml/min in 65 min, from 40 to 44% A with flow rate 0.3 to 0.7 ml/min in 70 min, 44% A with flow rate 0.7 to 1 ml/min for 10 min duration, solvent A changed from 44% to 58% with flow rate 1 ml/min for 5 min, 58 to 70% A in 98 min at constant flow rate 1 ml/ min. The mobile phase composition back to initial condition (solvent A: solvent B: 10: 90) in 101 min and allowed to run for another 4 min, before the injection of another sample. Total analysis time per sample was 105 min. HPLC chromatograms were detected using a photo diode array Ultra-Violet (UV) detector at three different wavelengths (272, 280 and 310 nm) according to absorption maxima of analysed compounds. Each compound was identified by its retention time and by spiking with standards under the same conditions. The quantification of phenolic acids and flavonoids in the sample extracts were carried out by the measurement of the integrated peak area and the contents were calculated using the calibration curve by plotting peak area against concentration of the respective standard sample. For the preparation of standard stock solutions of twenty one phenolic acids and flavonoids like gallic acid, protocatechuic acid, gentisic acid, p-hydroxy benzoic acid, catechin, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferullic acid, sinapic acid, salicylic acid, naringin, rutin, ellagic acid, myricetin, quercetin, naringenin, apigenin and kaempferol were prepared in methanol at 10 μg/ml-1. All standard solutions were filtered through HPLC filter 0.45 mm membrane filter (Milipore).

Sample preparation for evaluation of antioxidant properties

For evaluating the antioxidant properties, the young leaf tissue (35 days old) of ten experimental accessions of A. hypochondriacus, grown at Crop Research and Seed Multiplication Farm (CRSMF), The University of Burdwan, Burdwan, West Bengal, India were collected and washed thoroughly with normal tap water followed by sterile distilled water. Then leaves were dried at 45°C for 48 hours in hot air oven. Leaves were crushed to powder using mixer grinder. Powder was stored in tight air container bottle. The dried leaf tissue was extracted with methanol or deionised distilled water (Milli Q grade) for analysis antioxidant properties. The supernatant were stored in refrigerator for their future use for the following phytochemical analysis.

Metal chelating property: For the estimation of metal chelating property of experimental plant tissue, the process of Lin et al. [25] was followed with slight modifications. Shortly, 1 ml water extract (extraction procedure described earlier) was added to a solution of 0.02 ml 2 mM ferrous chloride and 0.04 ml 5 Mm ferrozine. The mixture was vigorously shaken and incubated for 10 min. Absorbance was taken at 562 nm. Metal chelating activity was expressed as:

Activity (%): [Ac-As/Ac] × 100

Where, Ac=Absorbance of control, As=Absorbance of sample.

Reducing power: For the estimation of reducing power of experimental plant tissue the process of Lin et al (2009) [24] was followed with slight modifications shortly, 1 g of dry powder was extracted with 50 ml of distilled water at 70°C under reflux for 4 hours and then centrifuged for 3000 rpm for 10 min. 25 ml of supernatant was taken and added with 200 Mm sodium- phosphate buffer (pH 6.6) and 0.1% potassium ferricyanide. The mixture was incubated for 20 min at 50°C and then added with 0.25 ml 10% TCA. Subsequently the mixture was centrifuged at 3000 rpm for 10 min. Supernatant was collected and mixed with deionised water and 1% ferric chloride solution. The mixture was kept for 10 min and absorbance was taken at 700 nm. Reducing power was expressed as activity (%).

Activity (%)=[Ac-As/Ac] × 100

Where, Ac=Absorbance of control, As=Absorbance of sample respectively.

Hydroxyl radical scavenging activity (OH.)

Hydroxyl radical scavenging capacity of methanolic extract of leaf sample was determined according to the method of Jan et al. [26] with slight modifications. The assay mixture (sample diluted with phosphate buffer 10 mM, pH 7.41 ml of 2.8 mM 2-deoxy-ribose, 20 μM FeCl3 and 100 μM EDTA, 200 μM H2O2 and 300 μM ascorbic acid) was incubated at 37°C for 1 hour. Then 1 ml of 2.8% TCA, 1 ml of 1% TBA and 0.1 ml 50 mM NaOH were added. The reaction mixture was heated in a boiling water bath for 15 min. The absorbance was recorded at 532 nm.The hydroxyl radical scavenging capacity was calculated according to equation:

Activity (%)=[Ac-As/Ac] × 100

Where, Ac=Absorbance of control, As=Absorbance of sample respectively.

Anti-lipid peroxidation assay in linoleic acid system

For the estimation of anti-lipid peroxidation assay experimental plant tissue the process of Amabye [27] was followed with slight modifications. The antioxidant activity of the different accessions of A. hypochondriacus extracts were determined by measuring the oxidation of linoleic acid. 5 mg of Amaranthus spinosus extracts were added separately to a solution of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 10 mL of 0.2 M Sodium Phosphate buffer (pH=7). The mixture was made up to 25 mL with distilled water and incubated at 40°C up to 360 hours. Extent of oxidation was measured by peroxide value applying thiocyanate method. Briefly, 10 mL of ethanol (75% v/v), 0.2 mL of an aqueous solution of ammonium thiocyanate (30% w/v), 0.2 mL of sample solution and 0.2 mL of ferrous chloride (FeCl2) solution (20 mM in 3.5% HC1; v/v) added sequentially. After 3 min of stirring, the absorption was measured at 500 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc. and Tokyo, Japan). A negative control contained all reagents with exception of extracts. Synthetic antioxidants BHT (also we can use ascorbic acid) was used as positive control. The maximum per oxidation level was observed at 360 hours (15 days) in the sample that possesses no antioxidant component percent inhibition of linoleic acid oxidation was calculated with the following equation:

% inhibition of linoleic acid peroxidation=[1-Change in absorbance of treated sample/Change in absorbance of control sample] × 100

2,2-Diphenyl-1-picryl-hydrazyl (DPPH) assay

For the determination of antioxidant properties using a free radical scavenging assay of DPPH method the process of Shyu and Hwang was followed [28]. One ml tissue extract was taken and 3 ml DPPH (0.04 mg/ml ethanol) was added and mixed thoroughly at room temperature. The mixture was incubated for 30 min at room temperature. The absorbance was read at 517 nm after 30 min of initial mixing. The same concentration of methanol (6 ml) was used as the control. The total antioxidant capacity was expressed as % DPPH. Scavenged and calculated as:

% DPPH. Scavenged = [1-(AI-AJ/Ac)] × 100

Where, Ai=Sample+DPPH solution, Aj=Sample+ethanol, Ac=Ethanol+DPPH solution.

Statistical Analysis

For statistical analysis of data, standard error was calculated using three replicate of independent contents. All the determinations were performed in triplicates. Data were also analysed for Analysis of Variance (ANOVA) test. The means of the significant differences were separated using Fisher’s least significant test for difference at the 0.05 level of probability.

Results and Discussion

The DPPH radical scavenging assay has been widely used to assess the total antioxidant capacity of plant extracts and products. This method employs the reduction of DPPH in presence of an antioxidant (electron donor) bringing about a change in colour from purple to yellow and measured at wavelength 517 nm. Table 1 shows a comparative account of the scavenging effect of methanolic leaf extracts of ten promising accessions of A. hypochondriacus, which is in the order IC107144 > IC47434> IC95251 > IC42397 > IC94661 > IC95316 > EC42352 > EC146543 > IC95322 > IC95326.


Accession No. of Amaranthus hypochondriacus L.

Anti-lipid peroxidation (% inhibition g-1 dm)

Reducing Power (% g-1 dm)

Metal chelating activity (% g-1 dm)

OH. Radical scavenging property (% g-1 dm)
Total Antioxidant capacity (DPPH % Radical scavenged g-1 dm)
EC42352
67.54 ± 0.51h
8.69 ± 0.12f
191.22 ± 1.05d
37.86 ± 0.24e
29.0 ± 1.05c
IC47434
482.20 ± 0.96b
27.04 ± 0.18a
286.81 ± 1.80b
64.71 ± 0.75b
33.8 ± 0.60a
IC94661
431.57 ± 1.38c
18.87 ± 0.24c
244.37 ± 2.16c
22.28 ± 0.18f
29.5 ± 0.90c
IC95251
425.14 ± 1.23c
13.20 ± 0.18d
152.16 ± 0.96g
41.68 ± 0.45d
31.4 ± 1.23b
IC95316
170.01 ± 0.63f
14.02 ± 0.15c
179.19 ± 1.38f
40.18 ± 0.60d
28.8 ± 0.96c
IC95322
108.50 ± 0.24g
16.72 ± 0.21d
138.56 ± 1.56e
41.50 ± 0.90a
27.7 ± 1.50d
IC95326
27.96 ± 1.05i
22.97 ± 0.24b
179.11 ± 2.31f
44.96 ± 0.75c
27.4 ± 1.20d
IC107144
502.11 ± 0.81a
28.79 ± 0.24a
295.57 ± 2.40a
90.23 ± 2.40a
34.2 ± 0.78a
EC146543
317.28 ± 0.66d
6.71 ± 0.03g
284.66 ± 2.10b
41.53 ± 1.20d
29.0 ± 0.90c
IC42397
295.98 ± 0.99e
13.10 ± 0.06e
164.58 ± 0.66e
67.72 ± 1.50b
30.5 ± 1.47c

Table 1: Biomarkers of Antioxidant potential of foliar hydro-ethanolic extracts of ten different experimental accessions of Amaranthus hypochondriacus L. assessed in terms of anti-lipid peroxidation property, reducing power, metal chelating activity and hydroxyl radical scavenging properties. Results are mean of three replicates ± standard deviation. Different super indexes indicate significant differences within a column, compared by Fishers least significant difference test (p<0.05).

Hydroxyl radical (OH.) is the most potent ROS and is capable of instigating oxidative determination of almost every important biomolecules. This scavenging hydroxyl radical is extremely important for protection of biological systems. In this study, methanolic leaf extracts of all the experimental accessions exhibit good OH radical scavenging property. Among the accessions, the accession no. IC107144, IC47434 and IC95251 are among the best three germplasms having OH∙ radical scavenging properties, corroborating well the data of total antioxidant property (DPPH∙ radical scavenging ability) of leaf extracts of the experimental accessions (Table1).

Table 1 also shows the reducing power of the experimental accessions by measuring the Fe3⁺ to Fe2⁺ transitions in presence of foliar extracts. Reducing power simply reflects the antioxidant function either by donating electrons or by forming radical chain breaking reaction. Other study showed the highest reducing power of methanolic leaf extract for the accessions IC107144 and IC47434 substantiating well with the data of total antioxidant capacity and OH∙ radical scavenging properties.

Transition metal ions, particularly Fe stimulate Fenton reaction and accelerate lipid peroxidation through conversion of hydroperoxides into alkyl and peroxyl radicals and hence perpetuate further the chain reaction of membrane lipid peroxidation. According to our result, the methanolic leaf extract of all the experimental accessions of A. hypochondriacus exhibit metal chelating property. But when compared, the leaf extracts of IC107144 and IC47434 possess significantly higher metal chelating property followed by IC95251 and others, corroborating again our earlier data of antioxidant properties of leaf samples i.e, reducing power, OH∙ radical scavenging properties and DPPH radical scavenging properties (Table 1).

Anti-lipid peroxidation of experimental plant sample was determined by inducing oxidation of linoleic acid as model system. Linoleic acid was incubated in oxidizing atmosphere with or without plant sample and subsequently the oxidizing value was measured by estimating the peroxide value applying thiocyanate method. The result of anti-lipid peroxidation assay for the leaf extracts of experimental accessions showed highest inhibition percentage for the accession IC107144 and IC47434 followed by others. So, like other attributes of antioxidant potential tested for the foliar extracts of different accession of A. hypochondriacus (OH∙ radical scavenging properties and DPPH radical scavenging properties, metal chelating property, reducing power), anti-lipid peroxidation property strongly corroborate the fact that the accession no IC107144 and IC47434 are the two lead germplasms compared to others when antioxidant property of the leaf extracts were considered.

Phenolics constitute diverse range of chemicals possessing at least one aromatic ring with -OH or other sub-constituent. RP-HPLC is the most sensitive and widely used techniques for identification of plant phenolic compounds. Because of the huge structural diversity of this class of secondary metabolites having antioxidant properties, it is entirely difficult to have a complete qualitative profiling of this class of compounds. So, we have targeted a major category of phenolic compounds with antioxidant potential for one of the lead germplasm of the experimental accession i.e. IC107144. The chromatographic separation of Retention Time (Rt) of gallic acid (Rt 7.69), protocatechuric acid (Rt 17.56), gentesic acid (Rt 30.88), p-hydroxy benzoic acid (Rt 36.76) catechin (Rt 40.50), chlorogenic acid (Rt 43.37), vanillic acid (Rt 45.58), caffeic acid (Rt 47.05), syringic acid (Rt 49.17), p-coumaric acid (Rt 55.27), ferulic acid (Rt 57.88), sinapic acid (Rt 62.66), naringin (Rt 70.78), rutin (Rt 72.40), ellagic acid (Rt 75.41) myricetin (Rt 77.90), quercetin (Rt 88.14), naringenin (Rt 92.76), apigenin (Rt 93.60) and kaemmpferol (Rt 99.42) are shown in Figure 1. The content of phenolic acids or flavonoids from hydro-ethanolic leaf extract of the lead experimental accession of A. hypochondriacus was estimated from the corresponding calibration curve and shown in Table 2. The RP-HPLC result based on computation against the Rt of standards revealed significant accumulation of the eight flavonoids and some phenolic acids like naringinin, rutin, myreicetin, quercetin, naringenin, apigenin, protocatechuric acid and p-hydroxy benzoic acid in the accession IC107144 (Figure 2). So, the red accession exhibited properties of significant accumulation of individual pharmacognosically important flavonoids, corroborating strongly the different attributes of antioxidant properties (OH∙ radical scavenging property, DPPH radical scavenging property, metal chelating property, reducing power and anti-lipid peroxidation property).

Figure 1

No.
Ret. Time
(Min)

Peak Name
Height
(mAU)
Area
(mAU* min)
Rel. Area
(%)
Amount
(μg/mL)

Type
1
7.69
Gallic acid
163.785
33.857
3.82
48.850
BMB
2
17.56
Protocatechuic acid
58.400
23.548
2.66
49.258
BMB
3
30.68
Gentisic acid
4.055
2.896
0.33
49.778
BMB
4
36.76
p-Hydroxy benzoic acid
59.882
31.371
3.54
49.881
BMB
5
40.50
Catechin
46.048
12.226
1.38
52.870
BMB
6
43.37
Chlorogenic acid
73.857
18.453
2.08
49.977
BMB
7
45.58
Vanillic acid
88.676
25.905
2.93
49.783
BMB
8
47.05
Caffeic acid
139.343
36.540
4.13
49.724
BMB
9
49.17
Syringic acid
175.276
46.144
5.21
54.377
BMB
10
55.27
p-Coumaric acid
323.326
85.974
9.71
50.205
MB
11
57.88
Ferullic acid
171.837
62.768
11.09
49.455
MB
12
62.66
Sinapic acid
327.629
227.163
25.66
49.961
BMB
13
69.53
Salicylic acid
22.048
11.438
1.29
48.058
BM
14
70.78
Naringin
62.808
29.490
3.33
53.243
M
15
72.40
Rutin
61.731
21.912
2.48
53.201
MB
16
75.41
Ellagic acid
76.632
23.457
2.65
45.058
BMB
17
77.90
Myricetin
97.082
30.853
3.49
49.223
BMB
18
88.14
Quercetin
153.098
25.228
2.85
44.642
BMB
19
92.76
Naringenin
107.321
20.963
2.37
44.964
BMB
20
93.60
Apigenin
137.405
27.878
3.15
44.795
BMB
21
99.42
Kaempferol
201.389
54.442
6.15
61.234
BM

Figure 1

Phenolic Acids and Flavonoids
Quantitative Amount (μg g-1 d.m.)
Gallic acid
0.0146436
Protocatechoic acid
0.3650183
Gentisic acid
1.009528
p-Hydroxy benzoic acid
0.11782166
Catechin
0.227721
Chlorogenic acid
0.241036
Caffeic acid
1.52964133
Syringic acid
0.774798
p-Coumaric acid
0.5439006
Ferullic acid
1.318666
Sinapic acid
0.297373
Naringin
0.073884
Rutin
8.49248
Ellagic acid
0.239743
Myricetin
0.9355766
Quercetin
0.449808
Naringenin
0.069234
Apigenin
0.50395
Kaempferol
0.9475876

Table 2: RP-HPLC based comparative variation of pharmacognosically important flavonoids and phenolic acids in the lead accession of A. hypochondriacus (accession no. IC107144).

Figure 2

No.
Ret. Time
(Min)

Peak Name
Height
(mAU)
Area
(mAU* min)
Rel. Area
(%)
Amount
(μg/mL)

Type
1
7.54
Gallic acid
2.017
1.218
0.07
1.757
BMB
2
17.13
Protocatechuic acid
26.278
20.940
1.21
43.802
bMB
3
30.07
Gentisic acid
6.646
7.047
0.41
121.143
MB
4
40.88
Catechin
12.974
6.319
0.37
27.327
BMb
5
44.03
Chlorogenic acid
32.239
10.680
0.62
28.924
M
6
46.76
Caffeic acid
404.862
134.889
7.80
183.557
BM
7
48.97
Syringic acid
256.738
78.899
4.56
92.976
M
8
55.59
p-Coumaric acid
289.721
111.769
6.46
65.268
M
9
57.51
Ferullic acid
531.655
200.838
11.61
158.240
MB
10
61.72
Sinapic acid
20.428
16.225
0.94
3.568
BMb
11
70.42
Naringin
7.821
4.911
0.28
8.866
bMB
12
71.86
Rutin
819.035
419.734
24.26
1019.098
BM
13
75.89
Ellagic acid
23.946
14.977
0.87
28.769
BM
14
77.10
Myricetin
17.832
7.037
0.41
11.227
MB
15
89.01
Quercetin
117.430
30.503
1.76
53.977
BMb
16
92.91
Naringenin
6.878
3.873
0.22
8.308
bMB
17
93.88
Apigenin
10.266
3.764
0.22
6.047
BMB
18
100.63
Kaempferol
297.742
88.026
5.09
113.711
BM

Figure 2

GC-MS method was employed for the ethanolic extract of young leaf tissue of lead accession no IC107144 for testing availability of hydroxyl rich phytochemical constituents. The acidic fraction was silyated and subjected to GC-MS investigation. It is evident from GC-MS spectra (Figure 3) that all fractions have a complex chemical composition. GC-MS data identified fifty four compounds from leaf extracts of accession no IC107144 based on library data (NIST and WILEY) of corresponding compounds. The ethanolic leaf extract of the lead experimental accessions IC107144 of A. hypochondriacus showed 17 major phenolic constituents as: 4H-pyran-4-one, 2-hydroxy (peak area 0.12%), /4H-pyran-4- one,2,3-dihydro-3,5-(peak area 1.68%)/, 2-methoxy-4-vinyl phenol (peak area 2%)/, 2-Furan carbamaldehyde, 5-(hydroxyl) (peak area 0.84%)/, Stevioside (peak area 0.33%)/, Napthalene 1,2-dihydro- 2,5,8-trimethyl (peak area 0.17%)/, 2,5,5-trimethyl-3-hexan-2-ol (peak area 0.19%) /4-(3-Hydroxy-2,2,6-trimethyl1-7-ol (peak area 0.10%)/, 2-Cyclohexene-1-one-4-(3-hydroxy, (peak area 0.27%)/, 4-(1,5-Dihydroxy-2,6-6 trimethyl (peak area.09%)/, 2-hexadecan- 1-ol,3,7,11,15, (peak area 2%)/, Octanamide, N-(2-hydroxyl), (peak area 0.16%)/, 1- hydroxyl-2,2,6,6-tetramethyl (peak area 0.42%)/, Hexadecanoic acid, 2-hydroxy-1 (2.95%)/, Stigmasterol-7-en-3-ol (1.81%)/, Gama-tocopherol (peak area 0.47%)/, Alpha-tocopherol (peak area 4.49%) and 4H-pyran-4-one, 2 hydroxy (peak area 0.07%)/, 4H-pyran-4-one, 2,3-dihydro-3,5-(peak area 0.17%)/, 3-heptanol (peak area 0.03%)/, 2-methoxy-4-vinyl phenol (peak area 0.35%)/, 1-tridecene (peak area 0.03%)/, Benzaldehyde,2-hydroxy- 1-propenyl)-2 (peak area 0.06%)/, 4-(1E)-3-hydroxyl-1-propenyl)-2 (peak area 0.06%)/, 13-Heptadecyn-1-ol (peak area 0.06%)/, Phytol isomer (peak area 17.7%)/, Hexadecanoic acid, 2-hydroxyl (peak area 0.05%)/, Cycloheptadecanol (peak area 0.68%)/, Hexadecanoic acid, 2-hydroxyl-1-(1,1,6-10-Dodecatrien-3-ol, 3 (peak area 3.82%)/, Gama-tocopherol (peak area 0.27%)/, Gama-tocopherol-beta-Dmannose (peak area 5.46%) respectively. So, the availability of wide diversity of OH-rich compounds further corroborates the significantly better antioxidant potential among the ten experimental accessions.

Figure 3

Figure 3

The etiology of all most all the degenerative diseases in one way or other are mediated by ROS or oxidative stress initiated by loss of redox homeostasis. Antioxidants are capable of restoring redox homeostasis either by delaying or by inhibiting over-production of ROS or by detoxifying them. Endogenous antioxidant, both enzymatic and non-enzymatic, cannot ensure rigorous control or complete protection against the oxidants under oxidative stress. So, supplementation of exogenous antioxidant goes up as nutritional supplements or pharmaceutical products containing active principles as antioxidant. Moreover, restriction of commonly used synthetic antioxidant for their non-target toxic effects has justified the demand for low cost natural source of antioxidants. A. hypochondriacus, a pseudo-cereal and a versatile multipurpose crop known for its excellent grain protein and other nutritional attributes is least exploited for its foliar nutritional status, particularly its antioxidant potential. Moreover, in spite of having huge genetic resources for A. hypochondriacus, a systematic exploration of nutritional attributes, particularly antioxidant in diverse land races are not explored.

So, here we preliminary screened the ten promising accessions of A. hypochondriacus for their foliar antioxidant potential through assessment of important antioxidant related attributes and projected them as biomarker for antioxidant potential. Due to completely complex nature of phytochemicals, it is not wise to use a single method for evaluating antioxidant potential of plant extract [29,30,31]. Therefore, we have used five different biomarkers like DPPH radical scavenging property, OH∙ radical scavenging property, metal chelating activity, reducing property and anti-lipid peroxidation property to validate the nature of leaf extracts from the experimental accessions of A. hypochondriacus in terms of desired antioxidant potential. Based on that, our result unequivocally identified a red accession IC107144 as the lead accession.

Several researchers reported phenolic acids, flavonoids, tannins and other hydroxyl rich compounds as the basis for the better metal chelating property, OH∙ radical scavenging property and reducing properties (21,26,31,32,33). Significantly better capacity of leaf extract of the red accession IC107144 to scavenge ROS and restore redox homeostasis is directly related to inhibition of lipid peroxidation and prevention of hydroxyl radical formation through metal chelation apart from chain breaking free radical reactions [25,26,29,33]. In fact, the total antioxidant capacity, OH radical scavenging property of the tissue extracts mainly reflects their nonenzymatic antioxidant activities [25,26,34-37].

The RP-HPLC procedure exploited in the present work provided excellent identification and qualification of 19 phenolic acids and flavonoids in leaves of the lead accession of A. hypochondriacus (IC107144). The experimental result not only confirmed the presence of all 19 phenolic acid and flavonoids but also revealed that the significant abundance of flavonoids and some phenolic acids corroborating well with the data of different facets of antioxidant potential tested and their prospective as antioxidant source with health benefit. The present study therefore has a potential application widely not only to quantify and compare antioxidant potential but also the availability of important flavonoids and phenolic acids in the lead accession of A. hypochondriacus.

The antioxidant property of Phenolic compounds generally resides with their radical scavenging ability in which they disrupt the free radical chain reaction (by H atom donation), resulting phenoxy radical, which subsequently got reduced (enzymatically or nonenzymatically) into parent compounds. Phenolic compounds may also cause metal chelation, particularly the transition metal ions, and reduce their availability required for generation of toxic ROS hydroxyl radicals, thereby can provide necessary components for radical scavenging properties and well corroborate with the present data of accession-specific variations of antioxidant properties [12,13,32,38,39,40]. It can also be possible that better radical scavenging property, reducing property, and antilipid peroxidation property of leaf extract of IC107144 may be due to –OH group present in phenolic compounds [21].

Further, to corroborate the data of outstanding antioxidant properties of the lead experimental germplasm of A. hypochondriacus L. (IC107144), GC-MS based method was employed for assessing the availability of –OH phenolic compounds present in the ethanolic leaf extract. GC-MS results indicated presence of several hydroxyl rich phytochemicals and some flavonoids from ethanolic extracts of leaves of the lead accession, which might be responsible for better antioxidant potentials of the accession no. IC107144 [21,32]. These accessions specific variations of antioxidant potential of A. hypochondriacus L. may depend on their origin and environmental regulation of redox metabolism [32,41,42]. Since phenolic compounds act as a free radical terminators exhibiting medicinal activity as well as important physiological function, the presence of these phytochemicals in the lead accession (IC107144) is a significant finding of the present study. The presence of those phytochemicals with -OH groups as well as their significantly high antioxidant attributes is also another significant finding in the present study.

Conclusion

Accession-specific variations of important antioxidant traits of foliar tissue of the seed amaranth, A. hypochondriacus L. were noticed and the lead germplasm was identified (IC107144). RP-HPLC provided sensitive tool for identification and quantification of several important phenolic acids and flavonoids for the lead red accession (IC107144). GC-MS results indicated presence of several hydroxylrich phytochemical constituents (phenolic compounds) in ethanolic leaf extracts of the lead experimental accession. Accession-specific comparative evaluation and identification of lead germplasm based on important biomarkers of antioxidant potential and the corresponding abundance of phenolic acids, flavonoids and phytochemicals with -OH groups in the lead accession of A. hypochondriacus L. is a significant finding of the present study.

Acknowledgement

Authors acknowledge UGC-CAS, Govt. of India to the Department of Botany, University of Burdwan for Research funding and facility to the Department [No. F.5-13/2012(SAPII)]. Special thanks are extended to the Director, National Bureau of Plant Genome Research, New Delhi, India for providing the seeds of the experimental accessions of A. hypochondriacus L. MA acknowledge thanks to the Director of Public Instructions, Higher Education Department, Government of West Bengal for kind cooperation in research work.