Nishi Srivastavaab,
Amit Srivastavaa,
S. Srivastavaa,
A. K. S. Rawat*a and
A. R. Khan*b
aPharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226001, India. E-mail: pharmacognosy1@rediffmail.com; Fax: +91-522-2207219; Tel: +91-522-2297816
bDepartment of Chemistry, Integral University, Lucknow-226001, India. E-mail: khanar70@yahoo.com; Tel: +91-522-2890730
First published on 2nd October 2014
The aim of the present communication is the development of validated HPTLC method for simultaneous separation, detection, comparative quantification of monomeric phenolic acids (MPAs), such as vanillic acid (VA), syringic acid (SYA), gallic acid (GA), protocatechuic acid (PCA) in Bergenia species viz. Bergenia ciliata (BC) and Bergenia stracheyi (BS) (Paashanbheda; family Saxifragraceae) and Kinetics studies on antioxidant activity of focused metabolites. The analyses were performed on HPTLC pre-coated silica gel 60F254 plates with optimized solvent system toluene:
ethyl acetate
:
formic acid (5
:
4
:
1 v/v/v) as mobile phase. Densitometric detection of MPAs was performed at 280 nm (λ max) wavelength. The contents of MPAs in both species were found (% in 10 mg ml−1) 0.007 ± 0.1–0.003 ± 0.4 (VA) (y = 3.326x − 1103, regression coefficient r = 0.998), 0.017 ± 0.4 − 0.002 ± 0.5 (SYA) (y = 3.410x − 1009, r = 0.998), 0.024 ± 0.2 − 0.012 ± 0.2 (GA) (y = 5.349x − 240.2, r = 0.999) and 0.027 ± 0.6 − 0.018 ± 0.2 (y = 3.6x − 461.5, r = 0.995). Quantitative variation was assumed as a result of samples collected from different altitudinal range. Two antioxidant assays DPPH and β-carotene were used kinetically in antioxidant potential assessment. Among both the species BC had higher DPPH antioxidant activity and antiradical kinetics than BS, MPAs and positive controls (TOCO), (BHT). Whilst in β-carotene assay highest antiradical activity was reported in PCA kinetically despite BHT than others. However, the deviation in CAA values of BC and BS extracts were very close to the PCA value. EC50 values, rate constant (k), rate of reaction (dx/dt), half-life and average life were also measured in both assays. On the basis of finding it can be concluded that the investigated MPAs were actively involved in antioxidant properties. The kinetic studies of MPAs revealed that H atom transfer from phenolic moieties to the ROS predicts the reactivity of antioxidants.
The pharmacological activity of extracts is dependent on the contents of active secondary metabolites in the plants. On varying the contents of secondary metabolites the activities also vary. Therefore, it is important to quantify the active secondary metabolites to find out the accurate pharmacological action of respective samples.
There are only few papers available on the analytical HPLC and HPTLC method development for the quantification of Bergenin and Gallic acid in different Bergenia species.10–12 No previous report is available on the simultaneous quantification of vanillic acid (VA), syringic acid (SYA), gallic acid (GA) and protocatechuic acid (PCA) in Bergenia species using high performance thin layer chromatography (HPTLC) (Fig. 1). Because of several advantages over other analytical methods, such as the rapidity, less amount of test sample and extremely limited solvent wastes, HPTLC has attracted massive interest as a most acceptable technique for the determination of pharmacologically interesting compounds in biological matrices such as plants and its different parts and even in formulations.13,14
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Fig. 1 Chemical structure of MPAs (VA = Vanillic acid, SYA = Syringic acid, GA = Gallic acid and PCA = Protocatechuic acid). |
Moreover, VA, SYA, GA and PCA were reported to possess various pharmacological effects, which may be closely correlated with its antioxidant activities.15,16 It has been well recognized that, several biochemical reactions involve the generation of ROS (reactive oxygen species) in human body. However, the balance between the generations of the diminution of ROS under normal conditions is controlled by antioxidant defense system. In case of certain pathological conditions, when ROS are not effectively eliminated by the antioxidant defense system, the dynamic balance between the generation and diminution of ROS is broken. The attack of excessive ROS and free radicals on carbohydrates, proteins, lipids, and DNA result into oxidative stress, which leads to various disorders and diseases.17 Antioxidants are compounds capable to either delay or inhibit the oxidation processes, which generate free radicals and reactive oxygen species. For the protection of bio-molecules against the attack of ROS, a no. of synthetic antioxidants such as 2- and 3-tert-butyl-4-methoxyphenol (i.e. butylated hydroxytoluene BHT, and tert-butylhydroquinone TBHQ) have been added to foodstuffs and also used for industrial processing in recent years, but because of their toxicity issues, their use is being questioned.18
To date, these synthetic antioxidants have been suspected of being harmful19 and cause severe side effects. Thus, in the recent years, search for natural antioxidants from plants is considerably focused. The plant derived antioxidants can be phenolic acid (flavonoids and tannins) nitrogen – containing compounds such as alkaloids, chlorophyll derivatives, amino acids, peptids), DL-α-Tocopherol acetate or ascorbic acid and its derivatives.20 Natural phenolic compounds are now proven as potent antioxidants, which quickly inhibit the generation of free ROS, compared to synthetic compounds. Therefore, plant extracts rich in polyphenolics are increasingly of interest to the food industry because they are capable to retard the oxidative degradation of lipids, and thereby improve the quality and nutritional value of the edible materials.
Consolidated comparative quantitative studies of MPAs using high performance thin layer chromatographic (HPTLC) and their antioxidant activity evaluation allows the analysts to determine the potency of each component within the total extract.21 Furthermore, it also allows the recovery of most active compounds and decides upon the best the development of technology for extraction, which enhances the quantity of potent compounds and to formulate products with these properties.22 In order to evaluate the antioxidant activity it is important to understand the mechanism of the reaction involved in scavenging for free radicals. According to DPPH assay the order of antioxidant activity (AA) is BC ∼ GA > PCA > SYA > BS > BHT ∼ TOCO ∼ VA of tested MPAs and extracts and in β-carotene the order of (AA) is BHT > PCA > TOCO > BC > GA > SYN > VA > BS. The results obtained from these two assays differ despite similar conditions used in the experiments. It seems important to notice that the compound, which is more active in DPPH assays, may not show the same potency in case of β-carotene assay. This contradiction can only be obvious, because the (AA) is not an inherent property of a particular compound, but depends on the nature of the free radical that is reacting with it. Free radical originated from hydrophilic reactions prefers polar compounds and those generated from lipophilic reactions like to be neutralized by non-polar antioxidants. Kinetic study is preferred to understand the order and mechanism of the reaction and it also helps in the estimation of different parameters required for the stability study of the compounds. Therefore, it was considered important to assess the comparative scavenging activity of each benzoic acid derivatives and extracts. For assessing the antioxidant activity DPPH and β-carotene assay were used.
Sample no. | Plant | State | Region explored | Collection stage | GPS information | Material |
---|---|---|---|---|---|---|
254021 | B. ciliata | Uttarakhand | Lansdowne | Pre-flowering | 9400 feet, N 31°03.116′ E 78°11.096′ | Whole plant |
262557 | B. stracheyi | Uttarakhand | Juda ka talab | Pre-flowering | 5400 feet, 29°50′N 78°41′E/29.83°N 78.68°E | Whole plant |
Parameters | VA | SYA | GA | PCA |
---|---|---|---|---|
Accuracy | 102.57 | 104.26 | 99.52 | 101.14 |
Rf value | 0.47 ± 0.02 | 0.43 ± 0.01 | 0.23 ± 0.01 | 0.38 ± 0.01 |
Regression equation | y = 3.326x − 1103 | y = 3.410x − 1009 | y = 5.349x − 240.2 | y = 3.6x − 461.5 |
Slope | 3.326 | 3.41 | 5.349 | 3.6 |
Intercept | 1103 | 1009 | 240.2 | 461.5 |
Linearity range | 1–6μg | 1–6μg | 1–6μg | 1–6μg |
95% Confidence limits of intercept | −518.0196009 | −94.16249112 | 267.4136872 | 1364.360849 |
Correlation coefficient (r) | 0.998 | 0.998 | 0.999 | 0.995 |
LOD | 510.7 | 778.06 | 275.23 | 602.83 |
LOQ | 1547.58 | 2357.76 | 834.03 | 4457.3 |
SE of intercept | 210.9533511 | 329.5067881 | 182.8382463 | 657.6350141 |
SD of intercept | 514.7261768 | 803.9965629 | 446.1253209 | 1604.629434 |
P-value | 0.006 | 0.03 | 0.0259173678 | 0.0521490957 |
MPA | Standard track r (s, m) | Sample track r (s, m) BC | Sample track r (s, m) BS | Standard track r (e, m) | Sample track r (e, m) BC | Sample track r (e, m) BS |
---|---|---|---|---|---|---|
VA | 0.999969 | 0.999263 | 0.998793 | 0.999942 | 0.997806 | 0.991686 |
SYA | 0.998756 | 0.998426 | 0.999878 | 0.996367 | 0.996143 | 0.999623 |
GA | 0.998256 | 0.998843 | 0.997241 | 0.996321 | 0.997944 | 0.987834 |
PCA | 0.998779 | 0.999037 | 0.998944 | 0.9973 | 0.997562 | 0.99383 |
S.No. | Sample | Extract (MeOH) | Applied Sample volume | % Content of MPAs in extract (10 mg ml−1) | |||
---|---|---|---|---|---|---|---|
10 mg ml−1; 10 μl | VA | SYA | GA | PCA | |||
1 | B. ciliata | Hydrolyzed extract | 10 μl | 0.007 ± 0.1 | 0.017 ± 0.4 | 0.024 ± 0.2 | 0.027 ± 0.6 |
2 | B. stracheyi | Hydrolyzed extract | 10 μl | 0.003 ± 0.4 | 0.002 ± 0.5 | 0.012 ± 0.2 | 0.018 ± 0.2 |
MPA | Amount present in BC in μg | Amount present in BS in μg | Amount added into sample | Theoretical value in BC | Theoretical value in BS | Average amount found in mixture of BC | Average amount found in mixture of BS | Average recovery in BC | Average recovery in BS |
---|---|---|---|---|---|---|---|---|---|
VA | 740 | 290 | 400 | 1140 | 690 | 1169.3 | 710.5 | 102.5701754 | 102.9710145 |
SYA | 1720 | 220 | 400 | 2120 | 620 | 2210.4 | 625.1 | 104.2641509 | 100.8225806 |
GA | 2410 | 1170 | 400 | 2810 | 1570 | 2796.6 | 1530.6 | 99.52313167 | 97.49044586 |
PCA | 2670 | 1820 | 400 | 3070 | 2220 | 3105 | 2201.4 | 101.1400651 | 99.16216216 |
MPA | Concentration (ng/spot) | Intraday | Interday | ||
---|---|---|---|---|---|
RSD% | Mean RSD% | RSD% | Mean RSD% | ||
VA | 4000–6000 | 2.65 | 99.80 ± 2.64 | 2.29 | 100.22 ± 2.29 |
SYA | 4000–6000 | 2.74 | 99.72 ± 2.73 | 2.75 | 100.7 ± 2.78 |
GA | 4000–6000 | 2.01 | 99.27 ± 1.99 | 1.37 | 100.28 ± 1.37 |
PCA | 4000–6000 | 4.71 | 99.82 ± 4.67 | 2.13 | 102.19 ± 1.37 |
Parameters | RSD% of peak area | |||
---|---|---|---|---|
(VA) | (SYA) | (GA) | (PCA) | |
a RSD = Relative standard deviation. | ||||
Time interval difference between spotting and plate development | 0.32 | 0.36 | 0.43 | 0.56 |
Mobile phase composition | 0.39 | 0.7 | 0.73 | 0.76 |
Time interval between drying and scanning | 0.37 | 0.5 | 0.67 | 0.78 |
The TPC varies from 24.2 to 179.1 μg GAE/mg extract (Fig. 6). The order of TPC descended in following order: BC > BS. Incidentally, BC with highest poly-phenolic contents also had higher amount of targeted compounds (i.e. VA, SYA, GA and PCA) as evident from HPTLC analysis.
The DPPH˙ radical has been widely used to estimate the free radical scavenging capacity of various antioxidants. The free radicals are scavenged by antioxidants that provide stability to the free radicals by electron or hydrogen donation. The un-reacted or remaining level of DPPH˙ in the reaction medium was calculated by using the following relation.
% of remaining DPPH˙ = 100 × [As517 nm (t = 30)/Ac517 nm] (eqn (1)), where As represents absorbance of sample at 517 nm (λ max) measured at (t = 30 min) and Ac represents absorbance of control at 517 nm (λ max) measured at (t = 0). It was observed that the % of the remaining DPPH· level linearly decreased with increasing B. ciliata and B. stracheyi concentrations to a certain level, and then leveled off. Along with extracts each identified MPAs (VA, SYA, GA and PCA) concentration effect on % of remaining DPPH˙ was also assessed. The effectiveness of the extracts of both the species of Bergenia in scavenging the free radicals was separately estimated as the half maximal effective concentration (EC50) (which refer to the concentration of drug, which induce a response half way between the baseline and maximum after a specified exposure time) of both the extracts in the reaction mixture that caused the decrease in the initial concentrations of DPPH˙ by 50%, denoted as EC50. EC50 values for the extracts of both the species of Bergenia and MPAs (VA, SYA and PCA) along with TOCO and BHT (used as positive control) are presented in (Table 7). The results of the investigated extracts, MPAs, TOCO and BHT, showed stronger free radical scavenging activity of BC extract over BS extracts, MPAs, TOCO and BHT. The effect of the extracts, MPAs (VA, SYA and PCA), positive control TOCO and BHT on the kinetics of free radical scavenging capacity for the investigated antioxidants is compared in (Fig. 7). In Fig. 8 the values of As 517 nm (t = x)-Ac517 nm are presented as the function of time and a concentrations of antioxidants, MPAs and positive control TOCO and BHT in the reaction mixture of amounting 0.1 mg mL−1. In (Fig. 7) Y-axis value As 517 nm (t = x)-Ac 517 nm refers to the concentrations of DPPH˙ scavenged at variable time interval (t = x). From Fig. 5, it is clear that in the presence of extracts of BC extract rapid initial decrease of DPPH˙ concentrations are followed by slow gradual disappearance of DPPH˙. Antioxidants quench the free radicals by two major mechanisms: by hydrogen atom transfer or via electron transfer that may also occur in parallel.26 However, the end result is the same regardless of the mechanism, but the kinetics differ.27 The contribution of the particular mechanism depends upon the compound involved (Fig. 9). DPPH˙ quenching is considered to be mainly based on electron transfer mechanism whilst hydrogen atom transfer mechanism is marginal reaction pathway.28 The reaction was initiated with transfer of either electron or hydrogen atom from antioxidants to the free radicals. Because it is clear from Fig. 7 that there are significant variations between the slopes after the completion of the initial fast step that do not rank in the manner as the EC50 values do. These variations are related to the role of slow secondary reactions, which may be due to the dimerization or disproportionation of initially formed phenol-derived radicals. To analyze the first rapid step of DPPH˙ quenching, different kinetic models have been proposed.29,30 To study the dynamic behavior of the system being analyzed, some mathematical models proposed by Saguy and Karel, 1980 (ref. 31) were used. It has already been established that antioxidant acidity follows the first order kinetics.32
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Fig. 7 The dependence of Ac 517 nm–As 517 nm (t = x) on time of incubation at a BC and BS extract and MPAs concentrations in the reaction mixture of 0.1 mg ml−1. Symbols represent experimental values; curves are plotted according to the parameters from eqn (1). |
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Fig. 8 The dependence of Ac 470 nm–As 470 nm (t = x) on time of incubation at a BC and BS extract and MPAs concentrations in the reaction mixture of 1 mg ml−1. Symbols represent experimental values, curves are plotted according to the parameters from eqn (1). |
To evaluate the mechanism and time dose-response of antioxidants in this investigation, a general reaction rate equation for first order kinetics can be written as follow −dx/dt = kf (x)m (eqn (2)), where x representing the concentration of reactant at time t, k represents rate of reaction of order m. In the above equation m = 1 and rate constant k are calculated at different time intervals depicted in (Table 8) Half-life of each were also calculated using first order half-life equation t1/2 = 0.693/k. Half-life of any compound represents the time at which half of concentration remains. Similarly, average life of MPAs were also calculated using the equation τ = 1/k.
Rate constantD K (mean ± SD) (1 × 10−2 (min−1) | Rate of reactionD (average) | EC50D (t = 0.25,10,20,30 min) | Half LifeD t1/2 (mean) | Average lifeD (τ) (mean) | Rate constantβ K (mean ± SD) (1 × 10−3 (min−1) | Rate of reactionβ (average) | CAAβ | Half lifeβ t1/2 (mean) | Average lifeβ (τ) (mean) | |
---|---|---|---|---|---|---|---|---|---|---|
BC | 88.27 ± 0.05 | 0.144788 | 54.08277t=0.2 | 0.768055 | 1.108305 | 30.129218 ± 0.07 | 0.01697 | 0.856718 | 24.82295 | 35.81955 |
54.90861t=10 | ||||||||||
55.06106t=20 | ||||||||||
52.24576t=30 | ||||||||||
BS | 88.82 ± 0.03 | 0.021078 | 53.84259t=0.25 | 0.76422 | 1.102771 | 22.473847 ± 0.04 | 0.014521 | 0.764849 | 31.09774 | 44.87409 |
66.86047t=10 | ||||||||||
72.03846t=20 | ||||||||||
5.586592t=30 | ||||||||||
VA | 76.30 ± 0.05 | 0.027918 | 223t=0.25 | 0.863595 | 1.246168 | 35.6869547 ± 0.05 | 0.186336 | 0.822632 | 20.88828 | 30.14182 |
87.75t=10 | ||||||||||
41.18519t=20 | ||||||||||
151.6875t=30 | ||||||||||
SYA | 83.96 ± 0.06 | 0.190908 | 7.87879t=0.25 | 0.799937 | 1.154311 | 34.978968 ± 0.06 | 0.017452 | 0.829644 | 23.03764 | 33.24335 |
8.83268t=10 | ||||||||||
47.70115t=20 | ||||||||||
64.01786t=30 | ||||||||||
GA | 102.11 ± 0.05 | 0.057927 | 57.5t=0.25 | 0.681032 | 0.98273 | 34.964833 ± 0.05 | 0.016605 | 0.849656 | 23.41162 | 33.78299 |
194.7475t=10 | ||||||||||
41.79063t=0 | ||||||||||
43.31719t=30 | ||||||||||
PCA | 91.51 ± 0.07 | 0.110352 | 56.63452t=0.25 | 0.745764 | 1.076139 | 36.06857 ± 0.06 | 0.014859 | 0.913298 | 24.06178 | 34.72118 |
54.69366t=10 | ||||||||||
54.13303t=20 | ||||||||||
55.33425t=30 | ||||||||||
BHT | 108.73 ± 0.03 | 0.036225 | 83.33333t=0.25 | 0.645982 | 0.932153 | 25.405815 ± 0.05 | 0.009022 | 0.981896 | 28.90811 | 41.71444 |
53.97154t=10 | ||||||||||
56.96815t=20 | ||||||||||
58.56125t=30 | ||||||||||
TOCO | 103.51 ± 0.06 | 0.019808 | 89.57143t=0.25 | 0.673302 | 0.971576 | 29.313845 ± 0.04 | 0.017703 | 0.861469 | 25.81279 | 37.24789 |
80.30435t=10 | ||||||||||
109.2903t=20 | ||||||||||
87.16667t=30 |
The rate of reaction (Rs) was calculated at different time intervals t = 0–0.25 min (initial rate), t = 0.25–5 min (reaction propagation), t = 5–10 min (after the completion of initial step) and at t = 15, 20, 25 min and t = 30 min (at the end of the observation when the reactions are presumably completed)and are depicted in (Table 7). To find the accurate EC50, a graph (Fig. 5) was plotted in between % inhibition and concentrations at different intervals and positive correlation coefficient of linear equation showed the value (r2 => 0.9). EC50 values were calculated by taking mean of minimum base to maximum range on Y-axis to the X-axis.
To understand the Kinetics of antioxidant activity we plotted a graph between % inhibition and rate of reaction, which showed positive correlation in the case of DPPH˙ antioxidant activity. Higher the value of rate of reaction, more will be the activity. To date this relationship has not been reported in previous studies. In first order kinetics the rate of reaction is directly proportional to the concentration of reactants at time t. Similarly, proportional relations were observed in the % inhibition and concentration. This relation led into the correlation of % inhibition, which is proportional to the rate of reaction (Fig. 10). In DPPH˙ scavenging activity, the availability of proton is responsible for attaining the stability of free radicals. The decreasing absorbance value indicates the stability of the free radical achieved by proton donated by targeted samples. In other words, the de-colorization of sample solution shows the positive antioxidant activity. The higher the ability of the samples to decolorize the DPPH˙ solution the more the potency of the samples will be. The overall results of the kinetic study are summarized in (Table 7). The order of DPPH˙ antioxidant activity is BC ∼ GA > PCA > SYA > BS > BHT ∼ TOCO ∼ VA. Results of (Fig. 5) concentrations Verses time also support the above statements.
Similarly, kinetic approach was also used for assessing antioxidant activity of extracts, MPAs (VA, SYA, GA, PCA), positive control TOCO and BHT in β-carotene antioxidant assay. In plant and living system multiple phases in which lipids and water coexist with some emulsifier, therefore, it becomes important to study the antioxidant assay using a heterogeneous system or emulsion is also required. The antioxidant activity using emulsions are defined as β-carotene antioxidant assay. The emulsion system of linoleic acid was used to estimate the antioxidant activity of the extracts, MPAs and positive controls. The temperature of the reaction was maintained under 50 °C to avoid or minimize the formation of side products. In the reaction mixture free radicals (peroxy radicals-ROO−)were formed from the oxidation of linoleic acid that attack on β-carotene (target molecule) and result in rapid de-colorization of the reaction medium. The mechanism of de-colorization of β-carotene can be slowed down by the subsequent addition of antioxidants, which donates hydrogen atom to quench the free peroxy radicals by converting them into lipid derivatives RCOOH via the following mechanism.
ROO˙ + β-carotene → bleaching | (1) |
ROO˙+ AH → RCOOH + A | (2) |
The Kinetic profile of auto-oxidation of polyunsaturated fatty acid was evaluated using the observed data from β-carotene-assay. β-carotene was exposed to free peroxy radicals (eqn (1) and (2)) formed from the emulsion of linoleic acid in the presence of antioxidants i.e. extracts, MPAs and positive control TOCO and BHT. The Kinetic of β-carotene assay was assessed in the same way as in the case of DPPH˙ quenching using the same expression [−dx/dt = kf(x)m] m = 1 (eqn (2)). The value Ac 470 nm-As 470 nm (t = x) refer to the change in the concentrations of β-carotene, which was obtained by the measurement of the absorbance of the sample, As 470 nm (t = x) at t = 20, 40, 60, 80, 100 and 120 min. The curve was plotted between value Ac 470 nm-As 470 nm (t = x) as a function of time in (Fig. 8). The extracts concentrations used in emulsion were 1 mg ml−1. Similarly, DPPH˙ free radical scavenging kinetics, the mathematical model that most satisfactorily describes the time dependence of Ac 470 nm-As 470 nm (t = x) for extracts, MPAs, positive controls TOCO and BHT is the as a function of time.
The antioxidant activity coefficient (CAA) was calculated according to following (eqn (3))
CAA = 1−[AS470nm(t=0) − As470nm(t=120)/Ac470nm(t=0) − Ac470nm(t=120)]. | (3) |
In (eqn (3)) AS470nm (t=0) denotes the initial absorbance of the sample along with antioxidants at time = 0 and As470nm(t=120) denotes the absorbance of sample at t = 120 min. Similarly, Ac470nm(t=0) shows the absorbance of control at t = 0 and Ac470nm(t=120) shows the absorbance of control at t = 120 min. The results obtained from both assays were almost similar and extract BC was found to be more active over BS, MPAs, TOCO despite BHT. The following order of activity based on CAA was achieved BHT > PCA > TOCO > BC > GA > SYN > VA > BS. The higher the value of CAA, the higher the β-carotene bleaching activity will be. In contrast to the DPPH˙ antioxidant activity, the relation between inhibition and rate was not found to be positive as in β-carotene. The correlation coefficient was obtained from the graph of % inhibition and the rate showed the R2 = 0.87. It has already been noted in the DPPH˙ antioxidant activity that greater the capacity of de-colorization of the sample solution, the higher will be the activity whilst in the case of β-carotene the inverse of the DPPH˙ observation was found, the lower the capacity of de-colorization or the higher the color retention of the sample solution, the higher will be the antioxidant activity (Fig. 11).
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Fig. 11 Correlation of rate of reaction of β-carotene free radical scavenging activity with % inhibition. |
The need for the kinetic study was to resolve the problem of present time, use of single-time dose response of one commercial antioxidants as calibration curve to compute the equivalently antioxidant activity of sample was considered as common and incorrect practice because of the availability of computational applications that provide the adequate tools to work with different variables in non-linear models also. The study of dose-response at one single time and expecting to find linear relation often lead to unreliable values hiding the real aspects of the actual responses. Multiple times dependent dose response can be used to find linear regression curve and that can be used to describe the whole kinetic profile.
BC | Bergenia ciliate |
BS | Bergenia stracheyi |
MPAs | Monomeric phenolic acids |
VA | Vanillic acid |
SYA | Syringic acid |
GA | Gallic acid |
PCA | Protocatechuic acid |
ROS | Reactive oxygen species |
RNS | Reactive nitrogen species |
BHT | Butylated hydroxytoluene |
TOCO | α-Tocopherol acetate |
This journal is © The Royal Society of Chemistry 2014 |