Plant Foods Hum Nutr DOI 10.1007/s11130-016-0527-8
ORIGINAL PAPER
Acidic Potassium Permanganate Chemiluminescence for the Determination of Antioxidant Potential in Three Cultivars of Ocimum basilicum Shivani Srivastava 1,2 & Alok Adholeya 1 & Xavier A. Conlan 2 & David M. Cahill 2
# Springer Science+Business Media New York 2016
Abstract Ocimum basilicum, a member of the family Lamiaceae, is a rich source of polyphenolics that have antioxidant properties. The present study describes the development and application of an online HPLC-coupled acidic potassium permanganate chemiluminescence assay for the qualitative and quantitative assessment of antioxidants in three cultivars of O. basilicum grown under greenhouse conditions. The chemiluminescence based assay was found to be a sensitive and efficient method for assessment of total and individual compound antioxidant potential. Leaves, flowers and roots were found to be rich reserves of the antioxidant compounds which showed intense chemiluminescence signals. The polyphenolics such as rosmarinic, chicoric, caffeic, p-coumaric, m-coumaric and ferulic acids showed antioxidant activity. Further, rosmarinic acid was found to be the major antioxidant component in water-ethanol extracts. The highest levels of rosmarinic acid was found in the leaves and roots of cultivars Bholy green^ (14.37; 11.52 mM/100 g DW respectively) followed by Bred rubin^ (10.02; 10.75 mM/ 100 g DW respectively) and Bsubja^ (6.59; 4.97 mM/ Electronic supplementary material The online version of this article (doi:10.1007/s11130-016-0527-8) contains supplementary material, which is available to authorized users. * David M. Cahill [email protected]
1
TERI–Deakin Nanobiotechnology Centre, The Energy and Resources Institute (TERI), DS Block, India Habitat Centre, Lodhi Road, New Delhi 110003, India
2
Deakin University, Geelong, Australia. Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, (Waurn Ponds Campus), 75 Pigdons Road, Locked Bag 20000, Geelong, Victoria 3220, Australia
100 g DW respectively). The sensitivity, efficiency and ease of use of the chemiluminescence based assay should now be considered for its use as a primary method for the identification and quantification of antioxidants in plant extracts. Keywords Antioxidants . Chemiluminescence . Ocimum basilicum . Polyphenolics . Rosmarinic acid
Introduction Plant derived polyphenolics are a potential source of antioxidant molecules [1–3]. They have found application in the food, nutritional and pharmaceutical industries as a replacement to synthetically derived antioxidants and as scavengers of free radicals [4]. Extracts from species such as Rosmarinus officnalis (rosemary), Salvia halophila (salvia) and Origanum vulgare (oregano) that belong to the family Lamiaceae have been extensively studied for their antioxidant potential [for example 5–7]. In contrast, there are comparatively few reports on the antioxidant potential of the nutritious and medicinally important herb Ocimum basilicum (basil) [8–10]. Ocimum basilicum is an aromatic herb with high morphological, genetic and biochemical diversity [11, 12], thus for screening and selection of high antioxidant yielding cultivars a fast, sensitive and efficient analytical method is required. The determination of total antioxidant activity has conventionally been based on batch style methodologies such as the 2-diphenyl-1-picrylhydrazyl radical (DPPH•) assay and the 2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS•+) and ferric reducing antioxidant power (FRAP) assays [4, 13, 14]. Multiple assays are often required for the complete assessment of the total antioxidant activity of plants and food related items, however, these assays also fail
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to provide any information about individual compounds linked to antioxidant activity [15]. Furthermore, bioassayguided fractionation studies are also often used but they are slow, tedious and expensive to perform and are often associated with loss of antioxidant activity [15, 16]. To overcome the disadvantages of the above assays and approaches online HPLC-coupled antioxidant determination assays are an effective alternative. We have previously reported the use of online HPLCcoupled acidic potassium permanganate chemiluminescence assay and have found it to be an efficient alternative to DPPH• and ABTS•+ assays for the assessment of total and individual compound antioxidant potential [2, 4, 15]. The acidic potassium permanganate assay has demonstrated high selectivity, and stability and correlation with bioactivity in comparison to the other online assays [4, 17, 18]. Also, we have widely used online acidic potassium permanganate based assay for the determination of the antioxidant potential of a range of polyphenolics in wines, teas, juices, coffees and thyme [2, 4, 15, 17], but further exploration is still needed for its application on medicinally important herbs. The present study therefore aimed at the development and application of an online HPLC-coupled acidic potassium permanganate chemiluminescence assay for the qualitative and quantitative analysis of readily oxidisable polyphenolics in the leaves, flowers and roots of three different cultivars of O. basilicum grown under greenhouse conditions. The developed method was used to identify the cultivar with the highest levels of antioxidants, the levels of activity in different organs and which polyphenolics were responsible for the activity.
Chemicals and Reagents Ethanol, 85 % ortho-phosphoric acid (OPA, AR grade) and analytical grade sulphuric acid were obtained from Merck (Kilsyth, Australia) while HPLC grade methanol was obtained from BDH Chemicals (Poole, England). Potassium permanganate was obtained from Chem Supply (Gillman, Australia). Sodium polyphosphate and standards of 3- hydroxybenzoic, m-coumaric, p-coumaric, caffeic, ferulic, vanillic, chicoric and rosmarinic acids were obtained from Sigma Aldrich (Castle Hill, Australia). HPLC grade (Milli Q) water (18.2 MΩ) was prepared in house and filtered through 0.2 μm filter. The acidic potassium permanganate reagent (1 × 10−3 M) was prepared by dissolution of potassium permanganate in 1 % (m/v) sodium polyphosphate solution adjusted to pH 2 with sulphuric acid. Sample Preparation and Determination of Total Phenolics For the estimation of total phenolics, total and individual polyphenolic antioxidant content, the extraction method described by Srivastava et al. [19] was used. Briefly, 50 mg of the lyophilized plant materials and 60 % (v/v) ethanol in water were used and extraction was performed twice for 15 min using a sonicator (B3510E-DTH, Branson Ultrasonics, Danbury, USA) on a final volume of 25 mL. The extracts were filtered using syringe filters (Millipore Millex HN, 0.45 μm, Merck, Darmstadt, Germany) and taken for the total phenolic analysis and total and individual compound antioxidant potential analysis. For the determination of total phenolics, a modified Folin-Ciocalteau colorimetric assay [19] was used.
Materials and Methods
Determination of Total Antioxidant Potential
Plant Material and Harvesting
An acidic potassium permanganate chemiluminescence detection system coupled to an Agilent 1200 HPLC system was used for determination of total antioxidant potential. The HPLC system (without a column in place) was coupled with a Minipuls 3 peristaltic pump, bridged PVC tubing, and custom built luminometer (which functions similarly to a conventional flow injection analysis system). All samples were diluted 100-fold using deionized water before injection. 50 μL of the sample at a flow rate of 3 mL/min was merged with acidic potassium permangaate (1 × 10−3 M) and the peak area was recorded to determine total antioxidant potential relative to the oxidisable molecules present in the extract [19]. To test the chemiluminescence response generated by polyphenolics found in O. basilicum, eight polyphenolics (3hydroxybenzoic, m-coumaric, p-coumaric, caffeic, ferulic, vanillic, chicoric and rosmarinic acids) were prepared to a concentration of 1 mg/mL in 100 % methanol and were analyzed as described in the separation methodology section.
Seeds of three different cultivars of O. basilicum namely Bsubja^, Bholy green^ and Bred rubin^ were purchased from commercial seed suppliers in India and Australia (Bangalore Seed Company, Bangalore, Karnataka, India and Australian Seed, Shoalwater, Western Australia, Australia). Seedlings at the two leaf stage were transferred to a 5 kg black plastic pot containing the soil mixed with absorbent granules (TERRAGREEN, Greenscape Aeration Company, Atascadero, USA). Plants were grown and replicated under greenhouse conditions at 25–28 °C as previously described [19]. The plants were watered by filling the pot to the top (maximum capacity) on a daily basis. After 90 days, the plants were harvested and divided into leaf, flower and root. The segregated plant parts were lyophilized (Labconco lyophilizer, Kansas City, USA) at −94.3 °C and 141 KPa for 48 h and used for extraction of polyphenolics and further analysis.
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Separation Methodology and Post Column Detection System All samples were separated using an HPLC mobile phase based on HPLC grade water +0.1 % OPA; v/v in water (solvent A) and methanol +0.1 % OPA; v/v in methanol (solvent B). A gradient program similar to the one described by Srivastava et al. [19] was used with a flow rate of 1.0 mL/ min, injection volume of 20 μL and UV detection at 280 nm. Separations were performed on an Apollo TM C 1 8 (150 mm × 4.60 mm × 5 μm particle diameter) column. Post-column acidic potassium permanganate chemiluminescence signals were generated using an in house built luminometer [4].
Statistical Analysis All data was expressed in terms of mean ± SEM. Raw data was analyzed using a commercial statistical package (GraphPad Prism 6). One way analysis of variance with a Tukey’s HSD test of significance at p ≤ 0.05 was used to determine the differences between cultivars for total phenolics and total and individual antioxidant potential for the whole plants and for different plant organs.
Total Antioxidant Potential For the assessment of total antioxidant potential, an HPLC system coupled with a post-column chemiluminescence detection unit was modified without a chromatography column to model a flow injection system (Electronic supplementary material, Fig. 1). Conditions identified by McDermott et al. [4], were used in the present study as our analyte type and the separation system was the same except for the flow rate which was 3 mL/min. Samples were diluted 100-fold before injection for elimination of any possible interference due to pH or dissolved solids [21]. Total antioxidant potential studies showed that the leaves, flowers and roots were good reserves of antioxidant molecules (Fig. 1a). Roots of cultivar Bsubja^ were found to have 1.4-fold greater peak area than leaves while roots for the other two cultivars showed lower antioxidant potential than the leaves. Interestingly, for the extracts obtained from the flowers, a greater peak area was observed in Bsubja^ when compared to the other two cultivars. The total antioxidant potential studies showed that roots of Bsubja^ were a greater source of antioxidant molecules while the leaves of Bholy green^ and Bred rubin^ had higher antioxidant potential than their respective flowers and roots. No significant difference (p ≤ 0.05) was found between the three
Results and Discussion For the high throughput screening studies of complex plant matrices, HPLC with UV detection is a useful technique for the determination of the concentration of an analyte, however it fails to provide any information about its reactivity. Coupling HPLC with post column detection assays which can provide information about reactivity of a molecule adds a further dimension to the standard analyses. Among the three (DPPH • , ABTS•+ and acidic potassium permanganate) commonly used reagents for post column assays, acidic potassium permanganate chemiluminescence is the most sensitive and rapid assay for analysis and quantification of readily oxidisable polyphenolics and other related compounds because of the simplified instrumentation, much quicker reagent preparation and reagent stability [2, 20, 21]. Water-ethanol (60 % (v/v) ethanol in water) was used for extraction and chemiluminescence studies in the current study because ethanol is used industrially and is a generally recognized as safe (GRAS) solvent [22]. Similar to our study use of 60 % ethanol in water and 15 mins maceration for optimal yield of antioxidants has been reported by Lugato et al. [23] in Passiflora alata Curtis.
Fig. 1 Quantification of total antioxidant potential and total phenolic content in different plant organs of three cultivars of O. basilicum. a Total antioxidant potential expressed as peak area in leaves, flowers and roots of all cultivars b Total phenolics content expressed in GAE mg/g DW in leaves, flowers and roots of all cultivars. Data is represented as mean ± SEM of three replicates
Plant Foods Hum Nutr Fig. 3 Combined plots of the UV absorbance and acidic potassium permanganate chemiluminescence chromatograms of leaves (a), flowers (b) and roots (c) of Bsubja^ cultivar of O. basilicum. Peak numbers represent common peaks found in both signals. Peak no. 3 is chicoric acid and peak no. 6 is rosmarinic acid
Preliminary Individual Polyphenolic Antioxidant Potential Study
Fig. 2 Chemiluminescence response produced by eight polyphenolics by the developed online HPLC-coupled acidic potassium permanganate chemiluminescence assay. Response generated is expressed in terms of peak area. CA (caffeic acid), ChA (chicoric acid), FA (ferulic acid), mCoA (meta coumaric acid), pCoA (para coumaric acid), PhA (3 hydroxybenzoic acid), RA (rosmarinic acid) and VA (vanillic acid)
cultivars for total antioxidant potential content found in the whole plant.
Total Phenolic Content Folin’s test based on an oxidation/reduction reaction estimates the number of reactive phenolic species present in the extracts. Total phenolic contents found in the current study showed that all plant organs are equally good reserves of phenolic compounds [24] The leaves of all the cultivars showed the same total phenolic content (66.99 ± 6.61 to 85.53 ± 8.82 GAE mg/ g DW) (Fig. 1b). Interestingly, flowers and roots of Bsubja^ were found to have significantly higher phenolic contents (68.20 ± 5.54, 80.69 ± 4.58 GAE mg/g DW, respectively) compared with the other two cultivars. A significant difference (p ≤ 0.05) was found among all the cultivars for the total phenolic content in the whole plant with the highest found in Bsubja^ (219.22 ± 4.50 GAE mg/g DW) and the lowest in Bred rubin^ (167.12 ± 2.81 GAE mg/g DW). Moderate correlation was found between the total phenolic content and the total antioxidant potential (r = 0.528) indicating that Folin’s reagent may not be suitable as a single assay for correlating phenolic content with antioxidant activity. Further, the ability of Folin’s reagent to react with sugar and ascorbic acid in addition to polyphenolics found in plant extracts limits its use as a single assay for correlation studies [20, 25]. Table 1 Analytical figures of merit for the three polyphenolic compounds found in O. basilicum
Polyphenolic
Rosmarinic acid Chicoric acid Caffeic acid
Eight commonly found polyphenolics in O. basilicum were analyzed for their chemiluminescence response using a post column acidic potassium permanganate assay (Electronic supplementary material, Fig. 2) [26–28]. Six out of eight polyphenolics (Fig. 2) elicited a significant chemiluminescence signal response with the most intense signal observed for rosmarinic (14.8 min) followed by chicoric (11.7 min), caffeic (10.9 min), p-coumaric (13.2 min), ferulic (13.4 min) and mcoumaric (14.4 min) acids. On the contrary, an almost negligible response was found for 3-hydroxybenzoic acid (10.8 min) and vanillic acid (11.2 min). The results are consistent with those presented by Mc Dermott et al. [4] and Costin et al. [20] showing that complex polyphenolics (rosmarinic and chicoric acids) provide a higher chemiluminescence response in comparison to simpler polyphenolics such as vanillic acid. This observation can be also correlated with extended electron delocalization and increased radical stabilization found in condensed ring structures [20]. Del Bano et al. [5] have described the correlation between structure of phenolic compounds and their antioxidant potential and have stated that the presence of catechol groups in the aromatic ring are an important determinant. Based on this, the high antioxidant potential of rosmarinic acid can be attributed to the presence of two catechol rings conjugated with a carboxylic acid in its chemical structure [5, 19]. A positive chemiluminescence response was also observed for chicoric acid showing its potential as an antioxidant molecule. As rosmarinic, chicoric and caffeic acids produced the highest chemiluminescence signal intensities, we tested the sensitivity of the developed chemiluminescence assay for these three polyphenolics and determined their analytical figures of merit (Table 1). The signal response from rosmarinic acid and caffeic acid fits well with the high level of sensitivity that has been shown earlier by Costin et al. [20] and McDermott et al. [4].
Retention time (min)
14.8 11.7 10.9
UV detection
Chemiluminescence
R2
LOD (μM)
R2
LOD (μM)
0.9988 0.9900 0.9922
0.5 0.2 10.0
0.9984 0.9870 0.9985
0.75 0.10 5.0
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Fig. 4 Chemiluminescence profile of leaves, flowers and roots from Bholy green^ (a) and Bred rubin^ (b) cultivars of O. basilicum. Peak no. 3 is chicoric acid and peak no. 6 is rosmarinic acid
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Post Column Acidic Potassium Permanganate Assay for Determination of Individual Compound-Related Antioxidant Potential in O. basilicum Gulcin et al. [10] examined the antioxidant activity of water extracts and ethanolic extracts of O. basilicum and advocated the use of five different batch style methodologies to provide complete information about the antioxidant status of the O. basilicum extracts. While this approach is commendable, the use of different antioxidant detection methodologies is time intensive, difficult to perform for screening of large sample sizes and also fails to provide any information about individual compounds present in the extract [29]. These drawbacks can be largely overcome by the use of the acidic potassium permanganate chemiluminescence assay as shown in the study conducted here. HPLC coupled acidic potassium permanganate chemiluminescence assay was chosen in the current study for analysis of antioxidants in O. basilicum because it reacts with potential antioxidants and shows a strong correlation between the analytical signal response and bioactivity [4, 17, 18]. A reverse phase separation system with post column acidic potassium permanganate assay for the analysis of the antioxidant potential of individual compounds was devised (Electronic supplementary material; Fig. 3). Aqueous methanol with ophosphoric acid was used as the mobile phase for separation of antioxidants because it does not interfere with the chemiluminescence detection [4, 15]. Chromatograms generated with the aid of chemiluminescence detection of all plant parts across all the cultivars were found to contain a substantial number (peak no. 1 to 10) of antioxidant peaks (Figs. 3 and 4). Table 1 in the Electronic supplementary material shows for all samples in the current study peaks commonly identified by both UV and chemiluminescence detectors. Comparison of UV absorbance peaks for leaves, flowers and roots of cultivar Bsubja^ with their chemiluminescence signals (retention times 8.6, 9.0, 10.3, 11.7, 12.2, 13.6, 14.8 min) showed similar chromatographic profiles (Fig. 3). This observation indicates the presence of antioxidant activity for all peaks as observed in earlier studies [15, 17]. By comparison with commercial standards peak no. 3 and 6 were
Table 2 Determination of antioxidant content (mM/100 g DW) for rosmarinic and chicoric acids found in the cultivars of O. basilicum
Cultivar
Subja Holy green Red rubin
identified as chicoric and rosmarinic acids, respectively, in all samples. The chemiluminescence signals obtained for all the three samples in the cultivar Bholy green^ are shown in Fig. 4a. In total, five peaks were observed and peak nos. 1, 3 and 6 had retention times (8.6, 11.7 and 14.8 min respectively) similar to that found in the Bsubja^ cultivar. A distinct peak (no. 9) was found at a retention time of 13.1 min both in the leaf and root samples while there was a small peak observed in the flower extract. The chemiluminescence-based chromatogram of Bred rubin^ (Fig. 4b) also showed peak 1 and 6 as common to the other two cultivars. Additionally, a very small, unknown peak was observed at a retention time of 11.00 min (peak no. 10) in all samples of Bred rubin^. In the present study, rosmarinic acid was observed as the major antioxidant peak in all plant parts across all cultivars of O. basilicum. In Bholy green^ and Bred rubin^, leaves were found as the major reserve of rosmarinic acid. Rosmarinic acid contributed 64, 43 and 39 % respectively to the total antioxidant peak area in the leaves of Bholy green^, Bsubja^ and Bred rubin^ cultivars. This outcome highlights the utility of rosmarinic acid as a potential chemomarker for the selection of cultivars of O. basilicum for high antioxidant content. Many peaks were detected in all extracts of O. basilicum in the present study (Electronic supplementary material; Table 1), out of which only two targeted peaks were studied. A mass spectral analysis of the same extracts in future would be useful for identification of unknown peaks as identification of all peaks was beyond the scope of the present study. Quantification of Identified Polyphenolics- Rosmarinic and Chicoric Acids The antioxidant potential of the two polyphenolics was quantified with the aid of standards (Table 2). Leaves, flowers and roots of Bsubja^ and Bholy green^ showed the presence of both rosmarinic and chicoric acids. In Bred rubin^ chicoric acid was not detected while rosmarinic acid was found in all plant parts. The highest rosmarinic acid level was found in leaves and roots of Bholy green^ followed by Bred rubin^ and Bsubja^. Similar to rosmarinic acid, a higher chicoric acid
Rosmarinic acid (mM/100 g DW)
Chicoric acid (mM/100 g DW)
Leaves 6.59 ± 0.39c a
14.37 ± 0.37 10.02 ± 1.10b
Flowers
Roots
Leaves
Flowers
Roots
8.03 ± 1.47a 5.38 ± 0.14a 7.58 ± 0.68a
4.97 ± 0.10b 11.52 ± 1.05a 10.75 ± 0.47a
1.47 ± 0.19a 1.58 ± 0.22a Nil
0.52 ± 0.84a 0.26 ± 0.33b Nil
1.20 ± 0.21a 1.55 ± 0.05a Nil
Different letters indicate significant differences between cultivars for rosmarinic and chicoric acids content in different plant parts
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concentration was found in leaves and roots of Bholy green^ than Bsubja^. Compared to flowers of the other two cultivars significantly higher levels of rosmarinic and chicoric acids were found in the flowers of Bsubja^. Importantly the distribution of rosmarinic acid found here showed that its content is cultivar- and plant organ-dependent.
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Conclusion The acidic potassium permanganate chemiluminescence based assay was efficient for studying the antioxidant potential in three different cultivars of O. basilicum. The total antioxidant potential assay showed that all plant parts are potential reserves of antioxidant molecules. Roots were found to be rich in antioxidant polyphenolics and it is suggested that further exploration of this underutilized resource would be of some benefit. Rosmarinic acid was identified as the dominant antioxidant molecule in all plant parts in all the cultivars of O. basilicum and on the basis of the antioxidant potential of rosmarinic acid, ‘holy green’ was found to be the highest producing cultivar. The current study has thus opened up a new application of the acidic potassium permanganate chemiluminescence-based assay for screening plants for antioxidant bioactives, their rapid assessment and also selection of antioxidant rich cultivars. ABTS •+ 2, 2′- azinobis-(3-ethylbenzothioazoline-6sulphonic acid) radical cation, DPPH• 2, 2-diphenyl-1picrylhydrazyl radical, FRAP Ferric reducing antioxidant power, GAE Gallic acid equivalents, HPLC High performance liquid chromatography, LOD Limit of detection, OPA orthophosphoric acid, UV Ultraviolet
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11. Acknowledgments We duly acknowledge the assistance from Mr. Shailendra Kumar for managing the greenhouse experiments at TERI. Infrastructure support provided by TERI, India and Deakin University, Australia is also acknowledged. Deakin University provided a post graduate scholarship to SS. Compliance with Ethical Standards
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Conflict of Interest The authors declare that there is no conflict of interest. 14.
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ORIGINAL PAPER
Acidic Potassium Permanganate Chemiluminescence for the Determination of Antioxidant Potential in Three Cultivars of Ocimum basilicum Shivani Srivastava 1,2 & Alok Adholeya 1 & Xavier A. Conlan 2 & David M. Cahill 2
# Springer Science+Business Media New York 2016
Abstract Ocimum basilicum, a member of the family Lamiaceae, is a rich source of polyphenolics that have antioxidant properties. The present study describes the development and application of an online HPLC-coupled acidic potassium permanganate chemiluminescence assay for the qualitative and quantitative assessment of antioxidants in three cultivars of O. basilicum grown under greenhouse conditions. The chemiluminescence based assay was found to be a sensitive and efficient method for assessment of total and individual compound antioxidant potential. Leaves, flowers and roots were found to be rich reserves of the antioxidant compounds which showed intense chemiluminescence signals. The polyphenolics such as rosmarinic, chicoric, caffeic, p-coumaric, m-coumaric and ferulic acids showed antioxidant activity. Further, rosmarinic acid was found to be the major antioxidant component in water-ethanol extracts. The highest levels of rosmarinic acid was found in the leaves and roots of cultivars Bholy green^ (14.37; 11.52 mM/100 g DW respectively) followed by Bred rubin^ (10.02; 10.75 mM/ 100 g DW respectively) and Bsubja^ (6.59; 4.97 mM/ Electronic supplementary material The online version of this article (doi:10.1007/s11130-016-0527-8) contains supplementary material, which is available to authorized users. * David M. Cahill [email protected]
1
TERI–Deakin Nanobiotechnology Centre, The Energy and Resources Institute (TERI), DS Block, India Habitat Centre, Lodhi Road, New Delhi 110003, India
2
Deakin University, Geelong, Australia. Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, (Waurn Ponds Campus), 75 Pigdons Road, Locked Bag 20000, Geelong, Victoria 3220, Australia
100 g DW respectively). The sensitivity, efficiency and ease of use of the chemiluminescence based assay should now be considered for its use as a primary method for the identification and quantification of antioxidants in plant extracts. Keywords Antioxidants . Chemiluminescence . Ocimum basilicum . Polyphenolics . Rosmarinic acid
Introduction Plant derived polyphenolics are a potential source of antioxidant molecules [1–3]. They have found application in the food, nutritional and pharmaceutical industries as a replacement to synthetically derived antioxidants and as scavengers of free radicals [4]. Extracts from species such as Rosmarinus officnalis (rosemary), Salvia halophila (salvia) and Origanum vulgare (oregano) that belong to the family Lamiaceae have been extensively studied for their antioxidant potential [for example 5–7]. In contrast, there are comparatively few reports on the antioxidant potential of the nutritious and medicinally important herb Ocimum basilicum (basil) [8–10]. Ocimum basilicum is an aromatic herb with high morphological, genetic and biochemical diversity [11, 12], thus for screening and selection of high antioxidant yielding cultivars a fast, sensitive and efficient analytical method is required. The determination of total antioxidant activity has conventionally been based on batch style methodologies such as the 2-diphenyl-1-picrylhydrazyl radical (DPPH•) assay and the 2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS•+) and ferric reducing antioxidant power (FRAP) assays [4, 13, 14]. Multiple assays are often required for the complete assessment of the total antioxidant activity of plants and food related items, however, these assays also fail
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to provide any information about individual compounds linked to antioxidant activity [15]. Furthermore, bioassayguided fractionation studies are also often used but they are slow, tedious and expensive to perform and are often associated with loss of antioxidant activity [15, 16]. To overcome the disadvantages of the above assays and approaches online HPLC-coupled antioxidant determination assays are an effective alternative. We have previously reported the use of online HPLCcoupled acidic potassium permanganate chemiluminescence assay and have found it to be an efficient alternative to DPPH• and ABTS•+ assays for the assessment of total and individual compound antioxidant potential [2, 4, 15]. The acidic potassium permanganate assay has demonstrated high selectivity, and stability and correlation with bioactivity in comparison to the other online assays [4, 17, 18]. Also, we have widely used online acidic potassium permanganate based assay for the determination of the antioxidant potential of a range of polyphenolics in wines, teas, juices, coffees and thyme [2, 4, 15, 17], but further exploration is still needed for its application on medicinally important herbs. The present study therefore aimed at the development and application of an online HPLC-coupled acidic potassium permanganate chemiluminescence assay for the qualitative and quantitative analysis of readily oxidisable polyphenolics in the leaves, flowers and roots of three different cultivars of O. basilicum grown under greenhouse conditions. The developed method was used to identify the cultivar with the highest levels of antioxidants, the levels of activity in different organs and which polyphenolics were responsible for the activity.
Chemicals and Reagents Ethanol, 85 % ortho-phosphoric acid (OPA, AR grade) and analytical grade sulphuric acid were obtained from Merck (Kilsyth, Australia) while HPLC grade methanol was obtained from BDH Chemicals (Poole, England). Potassium permanganate was obtained from Chem Supply (Gillman, Australia). Sodium polyphosphate and standards of 3- hydroxybenzoic, m-coumaric, p-coumaric, caffeic, ferulic, vanillic, chicoric and rosmarinic acids were obtained from Sigma Aldrich (Castle Hill, Australia). HPLC grade (Milli Q) water (18.2 MΩ) was prepared in house and filtered through 0.2 μm filter. The acidic potassium permanganate reagent (1 × 10−3 M) was prepared by dissolution of potassium permanganate in 1 % (m/v) sodium polyphosphate solution adjusted to pH 2 with sulphuric acid. Sample Preparation and Determination of Total Phenolics For the estimation of total phenolics, total and individual polyphenolic antioxidant content, the extraction method described by Srivastava et al. [19] was used. Briefly, 50 mg of the lyophilized plant materials and 60 % (v/v) ethanol in water were used and extraction was performed twice for 15 min using a sonicator (B3510E-DTH, Branson Ultrasonics, Danbury, USA) on a final volume of 25 mL. The extracts were filtered using syringe filters (Millipore Millex HN, 0.45 μm, Merck, Darmstadt, Germany) and taken for the total phenolic analysis and total and individual compound antioxidant potential analysis. For the determination of total phenolics, a modified Folin-Ciocalteau colorimetric assay [19] was used.
Materials and Methods
Determination of Total Antioxidant Potential
Plant Material and Harvesting
An acidic potassium permanganate chemiluminescence detection system coupled to an Agilent 1200 HPLC system was used for determination of total antioxidant potential. The HPLC system (without a column in place) was coupled with a Minipuls 3 peristaltic pump, bridged PVC tubing, and custom built luminometer (which functions similarly to a conventional flow injection analysis system). All samples were diluted 100-fold using deionized water before injection. 50 μL of the sample at a flow rate of 3 mL/min was merged with acidic potassium permangaate (1 × 10−3 M) and the peak area was recorded to determine total antioxidant potential relative to the oxidisable molecules present in the extract [19]. To test the chemiluminescence response generated by polyphenolics found in O. basilicum, eight polyphenolics (3hydroxybenzoic, m-coumaric, p-coumaric, caffeic, ferulic, vanillic, chicoric and rosmarinic acids) were prepared to a concentration of 1 mg/mL in 100 % methanol and were analyzed as described in the separation methodology section.
Seeds of three different cultivars of O. basilicum namely Bsubja^, Bholy green^ and Bred rubin^ were purchased from commercial seed suppliers in India and Australia (Bangalore Seed Company, Bangalore, Karnataka, India and Australian Seed, Shoalwater, Western Australia, Australia). Seedlings at the two leaf stage were transferred to a 5 kg black plastic pot containing the soil mixed with absorbent granules (TERRAGREEN, Greenscape Aeration Company, Atascadero, USA). Plants were grown and replicated under greenhouse conditions at 25–28 °C as previously described [19]. The plants were watered by filling the pot to the top (maximum capacity) on a daily basis. After 90 days, the plants were harvested and divided into leaf, flower and root. The segregated plant parts were lyophilized (Labconco lyophilizer, Kansas City, USA) at −94.3 °C and 141 KPa for 48 h and used for extraction of polyphenolics and further analysis.
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Separation Methodology and Post Column Detection System All samples were separated using an HPLC mobile phase based on HPLC grade water +0.1 % OPA; v/v in water (solvent A) and methanol +0.1 % OPA; v/v in methanol (solvent B). A gradient program similar to the one described by Srivastava et al. [19] was used with a flow rate of 1.0 mL/ min, injection volume of 20 μL and UV detection at 280 nm. Separations were performed on an Apollo TM C 1 8 (150 mm × 4.60 mm × 5 μm particle diameter) column. Post-column acidic potassium permanganate chemiluminescence signals were generated using an in house built luminometer [4].
Statistical Analysis All data was expressed in terms of mean ± SEM. Raw data was analyzed using a commercial statistical package (GraphPad Prism 6). One way analysis of variance with a Tukey’s HSD test of significance at p ≤ 0.05 was used to determine the differences between cultivars for total phenolics and total and individual antioxidant potential for the whole plants and for different plant organs.
Total Antioxidant Potential For the assessment of total antioxidant potential, an HPLC system coupled with a post-column chemiluminescence detection unit was modified without a chromatography column to model a flow injection system (Electronic supplementary material, Fig. 1). Conditions identified by McDermott et al. [4], were used in the present study as our analyte type and the separation system was the same except for the flow rate which was 3 mL/min. Samples were diluted 100-fold before injection for elimination of any possible interference due to pH or dissolved solids [21]. Total antioxidant potential studies showed that the leaves, flowers and roots were good reserves of antioxidant molecules (Fig. 1a). Roots of cultivar Bsubja^ were found to have 1.4-fold greater peak area than leaves while roots for the other two cultivars showed lower antioxidant potential than the leaves. Interestingly, for the extracts obtained from the flowers, a greater peak area was observed in Bsubja^ when compared to the other two cultivars. The total antioxidant potential studies showed that roots of Bsubja^ were a greater source of antioxidant molecules while the leaves of Bholy green^ and Bred rubin^ had higher antioxidant potential than their respective flowers and roots. No significant difference (p ≤ 0.05) was found between the three
Results and Discussion For the high throughput screening studies of complex plant matrices, HPLC with UV detection is a useful technique for the determination of the concentration of an analyte, however it fails to provide any information about its reactivity. Coupling HPLC with post column detection assays which can provide information about reactivity of a molecule adds a further dimension to the standard analyses. Among the three (DPPH • , ABTS•+ and acidic potassium permanganate) commonly used reagents for post column assays, acidic potassium permanganate chemiluminescence is the most sensitive and rapid assay for analysis and quantification of readily oxidisable polyphenolics and other related compounds because of the simplified instrumentation, much quicker reagent preparation and reagent stability [2, 20, 21]. Water-ethanol (60 % (v/v) ethanol in water) was used for extraction and chemiluminescence studies in the current study because ethanol is used industrially and is a generally recognized as safe (GRAS) solvent [22]. Similar to our study use of 60 % ethanol in water and 15 mins maceration for optimal yield of antioxidants has been reported by Lugato et al. [23] in Passiflora alata Curtis.
Fig. 1 Quantification of total antioxidant potential and total phenolic content in different plant organs of three cultivars of O. basilicum. a Total antioxidant potential expressed as peak area in leaves, flowers and roots of all cultivars b Total phenolics content expressed in GAE mg/g DW in leaves, flowers and roots of all cultivars. Data is represented as mean ± SEM of three replicates
Plant Foods Hum Nutr Fig. 3 Combined plots of the UV absorbance and acidic potassium permanganate chemiluminescence chromatograms of leaves (a), flowers (b) and roots (c) of Bsubja^ cultivar of O. basilicum. Peak numbers represent common peaks found in both signals. Peak no. 3 is chicoric acid and peak no. 6 is rosmarinic acid
Preliminary Individual Polyphenolic Antioxidant Potential Study
Fig. 2 Chemiluminescence response produced by eight polyphenolics by the developed online HPLC-coupled acidic potassium permanganate chemiluminescence assay. Response generated is expressed in terms of peak area. CA (caffeic acid), ChA (chicoric acid), FA (ferulic acid), mCoA (meta coumaric acid), pCoA (para coumaric acid), PhA (3 hydroxybenzoic acid), RA (rosmarinic acid) and VA (vanillic acid)
cultivars for total antioxidant potential content found in the whole plant.
Total Phenolic Content Folin’s test based on an oxidation/reduction reaction estimates the number of reactive phenolic species present in the extracts. Total phenolic contents found in the current study showed that all plant organs are equally good reserves of phenolic compounds [24] The leaves of all the cultivars showed the same total phenolic content (66.99 ± 6.61 to 85.53 ± 8.82 GAE mg/ g DW) (Fig. 1b). Interestingly, flowers and roots of Bsubja^ were found to have significantly higher phenolic contents (68.20 ± 5.54, 80.69 ± 4.58 GAE mg/g DW, respectively) compared with the other two cultivars. A significant difference (p ≤ 0.05) was found among all the cultivars for the total phenolic content in the whole plant with the highest found in Bsubja^ (219.22 ± 4.50 GAE mg/g DW) and the lowest in Bred rubin^ (167.12 ± 2.81 GAE mg/g DW). Moderate correlation was found between the total phenolic content and the total antioxidant potential (r = 0.528) indicating that Folin’s reagent may not be suitable as a single assay for correlating phenolic content with antioxidant activity. Further, the ability of Folin’s reagent to react with sugar and ascorbic acid in addition to polyphenolics found in plant extracts limits its use as a single assay for correlation studies [20, 25]. Table 1 Analytical figures of merit for the three polyphenolic compounds found in O. basilicum
Polyphenolic
Rosmarinic acid Chicoric acid Caffeic acid
Eight commonly found polyphenolics in O. basilicum were analyzed for their chemiluminescence response using a post column acidic potassium permanganate assay (Electronic supplementary material, Fig. 2) [26–28]. Six out of eight polyphenolics (Fig. 2) elicited a significant chemiluminescence signal response with the most intense signal observed for rosmarinic (14.8 min) followed by chicoric (11.7 min), caffeic (10.9 min), p-coumaric (13.2 min), ferulic (13.4 min) and mcoumaric (14.4 min) acids. On the contrary, an almost negligible response was found for 3-hydroxybenzoic acid (10.8 min) and vanillic acid (11.2 min). The results are consistent with those presented by Mc Dermott et al. [4] and Costin et al. [20] showing that complex polyphenolics (rosmarinic and chicoric acids) provide a higher chemiluminescence response in comparison to simpler polyphenolics such as vanillic acid. This observation can be also correlated with extended electron delocalization and increased radical stabilization found in condensed ring structures [20]. Del Bano et al. [5] have described the correlation between structure of phenolic compounds and their antioxidant potential and have stated that the presence of catechol groups in the aromatic ring are an important determinant. Based on this, the high antioxidant potential of rosmarinic acid can be attributed to the presence of two catechol rings conjugated with a carboxylic acid in its chemical structure [5, 19]. A positive chemiluminescence response was also observed for chicoric acid showing its potential as an antioxidant molecule. As rosmarinic, chicoric and caffeic acids produced the highest chemiluminescence signal intensities, we tested the sensitivity of the developed chemiluminescence assay for these three polyphenolics and determined their analytical figures of merit (Table 1). The signal response from rosmarinic acid and caffeic acid fits well with the high level of sensitivity that has been shown earlier by Costin et al. [20] and McDermott et al. [4].
Retention time (min)
14.8 11.7 10.9
UV detection
Chemiluminescence
R2
LOD (μM)
R2
LOD (μM)
0.9988 0.9900 0.9922
0.5 0.2 10.0
0.9984 0.9870 0.9985
0.75 0.10 5.0
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Fig. 4 Chemiluminescence profile of leaves, flowers and roots from Bholy green^ (a) and Bred rubin^ (b) cultivars of O. basilicum. Peak no. 3 is chicoric acid and peak no. 6 is rosmarinic acid
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Post Column Acidic Potassium Permanganate Assay for Determination of Individual Compound-Related Antioxidant Potential in O. basilicum Gulcin et al. [10] examined the antioxidant activity of water extracts and ethanolic extracts of O. basilicum and advocated the use of five different batch style methodologies to provide complete information about the antioxidant status of the O. basilicum extracts. While this approach is commendable, the use of different antioxidant detection methodologies is time intensive, difficult to perform for screening of large sample sizes and also fails to provide any information about individual compounds present in the extract [29]. These drawbacks can be largely overcome by the use of the acidic potassium permanganate chemiluminescence assay as shown in the study conducted here. HPLC coupled acidic potassium permanganate chemiluminescence assay was chosen in the current study for analysis of antioxidants in O. basilicum because it reacts with potential antioxidants and shows a strong correlation between the analytical signal response and bioactivity [4, 17, 18]. A reverse phase separation system with post column acidic potassium permanganate assay for the analysis of the antioxidant potential of individual compounds was devised (Electronic supplementary material; Fig. 3). Aqueous methanol with ophosphoric acid was used as the mobile phase for separation of antioxidants because it does not interfere with the chemiluminescence detection [4, 15]. Chromatograms generated with the aid of chemiluminescence detection of all plant parts across all the cultivars were found to contain a substantial number (peak no. 1 to 10) of antioxidant peaks (Figs. 3 and 4). Table 1 in the Electronic supplementary material shows for all samples in the current study peaks commonly identified by both UV and chemiluminescence detectors. Comparison of UV absorbance peaks for leaves, flowers and roots of cultivar Bsubja^ with their chemiluminescence signals (retention times 8.6, 9.0, 10.3, 11.7, 12.2, 13.6, 14.8 min) showed similar chromatographic profiles (Fig. 3). This observation indicates the presence of antioxidant activity for all peaks as observed in earlier studies [15, 17]. By comparison with commercial standards peak no. 3 and 6 were
Table 2 Determination of antioxidant content (mM/100 g DW) for rosmarinic and chicoric acids found in the cultivars of O. basilicum
Cultivar
Subja Holy green Red rubin
identified as chicoric and rosmarinic acids, respectively, in all samples. The chemiluminescence signals obtained for all the three samples in the cultivar Bholy green^ are shown in Fig. 4a. In total, five peaks were observed and peak nos. 1, 3 and 6 had retention times (8.6, 11.7 and 14.8 min respectively) similar to that found in the Bsubja^ cultivar. A distinct peak (no. 9) was found at a retention time of 13.1 min both in the leaf and root samples while there was a small peak observed in the flower extract. The chemiluminescence-based chromatogram of Bred rubin^ (Fig. 4b) also showed peak 1 and 6 as common to the other two cultivars. Additionally, a very small, unknown peak was observed at a retention time of 11.00 min (peak no. 10) in all samples of Bred rubin^. In the present study, rosmarinic acid was observed as the major antioxidant peak in all plant parts across all cultivars of O. basilicum. In Bholy green^ and Bred rubin^, leaves were found as the major reserve of rosmarinic acid. Rosmarinic acid contributed 64, 43 and 39 % respectively to the total antioxidant peak area in the leaves of Bholy green^, Bsubja^ and Bred rubin^ cultivars. This outcome highlights the utility of rosmarinic acid as a potential chemomarker for the selection of cultivars of O. basilicum for high antioxidant content. Many peaks were detected in all extracts of O. basilicum in the present study (Electronic supplementary material; Table 1), out of which only two targeted peaks were studied. A mass spectral analysis of the same extracts in future would be useful for identification of unknown peaks as identification of all peaks was beyond the scope of the present study. Quantification of Identified Polyphenolics- Rosmarinic and Chicoric Acids The antioxidant potential of the two polyphenolics was quantified with the aid of standards (Table 2). Leaves, flowers and roots of Bsubja^ and Bholy green^ showed the presence of both rosmarinic and chicoric acids. In Bred rubin^ chicoric acid was not detected while rosmarinic acid was found in all plant parts. The highest rosmarinic acid level was found in leaves and roots of Bholy green^ followed by Bred rubin^ and Bsubja^. Similar to rosmarinic acid, a higher chicoric acid
Rosmarinic acid (mM/100 g DW)
Chicoric acid (mM/100 g DW)
Leaves 6.59 ± 0.39c a
14.37 ± 0.37 10.02 ± 1.10b
Flowers
Roots
Leaves
Flowers
Roots
8.03 ± 1.47a 5.38 ± 0.14a 7.58 ± 0.68a
4.97 ± 0.10b 11.52 ± 1.05a 10.75 ± 0.47a
1.47 ± 0.19a 1.58 ± 0.22a Nil
0.52 ± 0.84a 0.26 ± 0.33b Nil
1.20 ± 0.21a 1.55 ± 0.05a Nil
Different letters indicate significant differences between cultivars for rosmarinic and chicoric acids content in different plant parts
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concentration was found in leaves and roots of Bholy green^ than Bsubja^. Compared to flowers of the other two cultivars significantly higher levels of rosmarinic and chicoric acids were found in the flowers of Bsubja^. Importantly the distribution of rosmarinic acid found here showed that its content is cultivar- and plant organ-dependent.
3.
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Conclusion The acidic potassium permanganate chemiluminescence based assay was efficient for studying the antioxidant potential in three different cultivars of O. basilicum. The total antioxidant potential assay showed that all plant parts are potential reserves of antioxidant molecules. Roots were found to be rich in antioxidant polyphenolics and it is suggested that further exploration of this underutilized resource would be of some benefit. Rosmarinic acid was identified as the dominant antioxidant molecule in all plant parts in all the cultivars of O. basilicum and on the basis of the antioxidant potential of rosmarinic acid, ‘holy green’ was found to be the highest producing cultivar. The current study has thus opened up a new application of the acidic potassium permanganate chemiluminescence-based assay for screening plants for antioxidant bioactives, their rapid assessment and also selection of antioxidant rich cultivars. ABTS •+ 2, 2′- azinobis-(3-ethylbenzothioazoline-6sulphonic acid) radical cation, DPPH• 2, 2-diphenyl-1picrylhydrazyl radical, FRAP Ferric reducing antioxidant power, GAE Gallic acid equivalents, HPLC High performance liquid chromatography, LOD Limit of detection, OPA orthophosphoric acid, UV Ultraviolet
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11. Acknowledgments We duly acknowledge the assistance from Mr. Shailendra Kumar for managing the greenhouse experiments at TERI. Infrastructure support provided by TERI, India and Deakin University, Australia is also acknowledged. Deakin University provided a post graduate scholarship to SS. Compliance with Ethical Standards
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Conflict of Interest The authors declare that there is no conflict of interest. 14.
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