determination of total antioxidant content of centipeda cunninghamii

Determination of Total Antioxidant content of Centipeda Cunninghamii

Thomas Wu, Mitchell Low, Dr Cheang Khoo*

* School of Science and Health, University of Western Sydney, Campbelltown New South Wales, 2179, Australia9548-1575. [email protected]

Abstract: The interest in commercialising the powerful biological activity of polyphenols and flavonoids outlines the necessity of determining the content of Centipeda Cunninghamii. The total polyphenolic content was determined by using the Folin-Ciocalteu method while the total content of flavonoids was determined spectrophotmetrically using the aluminium chloride colorimetric assay. The total polyphenolic content was measured to be 10.082mg/g gallic acid equivalent (GAE) at the highest and 6.21mg/g GAE at the lowest. The highest flavonoid content was identified to be at 50.304mg/g quercitin equivalent (QE) and the lowest was 14.061mg/g QE. The present paper also shows the ratio of flavonoids to polyphenolics of each sample as well as a comparison to results obtained via HPLC method.

Keywords: Centipeda Cunninghamii, colorimetric, polyphenol, flavonoid

In recent times, the awareness of the importance of antioxidants in maintaining health and protection from oxidative stress has led to a dramatic increase in the development of functional foods and extraction of therapeutic compounds for commercialisation1. Antioxidants have a range of functional properties including the reduction in the effects of coronary heart disease, food preservation, stabilizers in fuels and lubricants1,2. Two types of antioxidants: polyphenols and flavonoids, will be discussed in this paper. Polyphenols are a class of plant metabolites that are characterised by one or more aromatic rings with at least one attached hydroxyl group3,4. There are at least 8000 known naturally occurring phenolics and half of these are flavonoids. Besides the functional properties stated above, phenolics also contain a wide range of biochemical activities including antimutagenic and anticarcinogenic4. Flavonoids are a subclass of phenolics and have different biological activities to other polyphenols. Their biochemical activities include antimicrobial, antiarthritic, antiangiogenic and protein kinase inhibition4. Due to their polyphenolic nature, polyphenols and flavonoids are capable of scavenging free radicals such as super oxide and hydroxyl radicals. It is because of this ability and the other biochemical activities that have made these class of antioxidants the target for commercialisation by pharmaceutical companies.Centipeda Cunninghamii, otherwise known as Old-Man's weed is a native Australian plant that grows along the Murray River and along the edge of receding billabongs5. Historically, it has been used by aborigines as a medicinal plant for treating wounds and infections6. More recently, it has been used in a range of cosmetic products such as skincare, deodorants and as a general anti-inflammatory and antioxidant5. The total phenolic and flavonoid content of the Centipeda Cunninghamii is reported in this paper. The total phenolic content was determined using the Folin-Ciocalteu (F-C) reagent colorimetric method. This assay relies on the principle of electron transfer between the phenolic compounds to the phomolybdic/phosphotungstic acid complexes of the F-C reagent in an alkaline environment to form a blue coloured complex which can then be determined using UV-vis spectrophotometry8. Gallic acid was used as the standard in this F-C assay as it is one of the more common standards used in most published literature4,7-10 as well as a host of other reasons such as satisfactory solubility, adequate stability, low price and the purity is easily obtainable and retained9. The total flavonoid content was determined using the Aluminium chloride colorimetric10. This assay relies on aluminium chloride forming acid stable complexes with the ketone groups and acid labile complexes with ortho-dihydroxyl groups of the flavonoids. These complexes can then be detected through UV-vis spectrophotometry. Five samples of Centipeda Cunninghamii will be tested via these colorimetric methods, 3 dry herbs and 2 liquid extracts.Experimental ProcedureTotal phenolic assayThe total phenolic content was determined using the Folin-Ciocalteu assay9. An aliquot (15L) of herbal extract or gallic acid standard solution (1.5, 3.125, 6.25, 12.5, 25, 75mg/mL) was added to an test tube containing 25mL of F-C reagent. The contents were vortexed for 10s and left to stand at room temperature for 2min. 2mL of 5%(w/v) sodium carbonate solution and 2mL of distilled water was added after 2min. This mixture was vortexed for another 10s and incubated at 45oC for 30min then cooled rapidly with ice. The absorbances were measured at 760nm and all samples were analysed in triplicates.

Total flavonoid assayTotal flavonoid content was determined using the aluminium chloride assay11.An aliquot (0.5mL) of sample extract or quercitin standard solution (3, 6, 12.5, 25, 50mg/mL) was added to a test tube containing 300L of (1:20 w/v) NaNO2. The contents were vortexed for 10s and left to stand at room temperature for 5min. 300L of AlCl3 (1:10 w/v), 4mL of 1M NaOH and 3.9mL of distilled H2O was added to the mixture. This was vortexed for 10s and the absorbance was measured at 460nm with all samples analysed in duplicates.

Results and DiscussionPolyphenolic Content

Figure 1: Gallic acid standard curve.

Table 1: Absorbance readings for polyphenolic test on herb and liquid samples. Absorbance for triplicate determinationsAverage RSD

Sample123

201 (dry herb)0.0630.005*0.1670.115 0.009

202 (dry herb)0.1390.06*0.0960.1175 0.004

203 (dry herb)0.1030.036*0.1420.1225 0.003

204 (liquid extract)0.079*0.1410.1610.151 0.002

205 (liquid extract)0.358*0.2370.2470.242 0.002

* These results were deemed to be outliers and skewed the results. These results were not included in the average. Table 2: Calculated concentration of polyphenols in mg/g of gallic acid equivalent.SampleConcentration from Graph (mg/mL)Concentration of Stock solution (mg/g)

201 (dry herb)2.3669.465

202 (dry herb)2.4189.671

203 (dry herb)2.52110.082

204 (liquid extract)3.1076.21


Gallic acid was used as a standard in determining the total polyphenolic content of centipeda cunninghamii. As it can be seen in Table 2, the final concentration of the stock solution between the 5 samples were fairly consistent with the exception of sample 204. Samples 201-203 were herbal samples in which it had to be submerged in methanol and sonicated to allow as much of the polyphenols to dissolve into solution. Samples 204 and 205 were liquid extract of the herb and could be used directly without further processing. Each sample were grown in different environments (i.e. different soil pH, sunlight exposure etc.) to determine which environment was optimal in producing the highest amount of polyphenols although for the purpose of this study, the environments for each sample was not known to prevent bias. Once the concentration of each sample was obtained, there were compared to the concentrations obtained via a HPLC method. Although a direct comparison cannot be performed as the HPLC method does not use gallic acid as a standard and it is more selective to each unique polyphenol, a general comparison can be made in terms of the approximation of concentrations between the two methods possibly resulting in the use of the simpler and faster colorimetric method instead of the HPLC method if the results were comparable. The results of the HPLC method for polyphenols and flavonoids can be found in appendix 1. From the results, it can be seen that the concentration for the dry herbs were comparable to that of the HPLC results while the concentration for the liquid extracts were significantly higher than that of the HPLC results. The single lower dry herb concentration (sample 201) of the colorimetric method could be explained by a few factors including the solvent (methanol) used was insufficient to dissolve all the polyphenols in the dry herb, or the polyphenols in the dry herb is no longer active or could have decomposed during the drying process or the solution (herb + methanol) was not left to stand long enough for all the polyphenols to diffuse and dissolve into the methanol. Flavonoid Content

Figure 2: The quercitin standard curve.Table 3: Absorbance readings for flavonoid test on herb and liquid samples.Absorbance for duplicate determinations

Sample12Average RSD

201 (dry herb)0.740.8390.790 0.055

202 (dry herb)0.7080.7360.722 0.014

203 (dry herb)0.9420.8790.911 0.041

204 (liquid extract)0.2220.2870.255 0.012

205 (liquid extract)0.2040.3490.277 0.028

Table 4: Calculated concentration of flavonoids in mg/g of quercetin equivalent.SampleConcentration From Graph (mg/mL)Concentration from Stock Solution (mg/g)*

201 (dry herb)43.61943.619

202 (dry herb)39.90039.900

203 (dry herb)50.30450.304



*Since no dilutions were performed on the stock solution, concentration is the same as that from the graph.In determining the flavonoid content of centipeda cunninghamii, quercitin was used as a standard in producing the calibration curve. As seen in table 4, the herbal samples 201-203 had flavonoid concentration ranging from 40-50 mg/g of quercitin. Liquid extracts 204 and 205 had flavonoid concentrations of 14 and 15 mg/g of quercitin equivalent. Both the dry herb flavonoid concentrations and the liquid extract concentrations are significantly higher than that of the HPLC results. Several possible explanations for the consistent and significantly higher flavonoid content includes the possibility of other substances in the herb that also absorbed light with this method causing an overall apparent high levels of flavonoid concentration.

Table 5: Concentration of polyphenols (P) and flavonoids (F) with F/P ratio.SampleConcentration of polyphenol (mg/g GAE)Concentration of Flavonoid (mg/g QE)Flavonoids/Polyphenol(F/P ratio)

201 (dry herb)9.46543.6194.608

202 (dry herb)9.67139.9004.125

203 (dry herb)10.08250.3044.989

204 (liquid extract)6.2114.0612.264

205 (liquid extract)9.95915.2761.534

Table 6: Comparison of Colorimetric results to HPLC resultsSamplePolyphenols%differenceFlavonoids% difference

ColorimetricHPLC(-HPLC)ColorimetricHPLC(- HPLC)

201 (dry herb)9.46512.7-34.1843.6193.8+1047.87

202 (dry herb)9.6719.0+7.46

39.9003.5+1040

203 (dry herb)10.08210.0+0.8250.3043.0+1576

204 (liquid extract)6.211.70+265.2914.0610.17+8171.18

205 (liquid extract)9.9591.61+518.5715.2760.17+8885.89

When determining the validity of the results, the concentration of polyphenols and flavonoids of the plant centipeda cunninghamii needs to be compared together. From the table 5 it can be seen that the ratio of flavonoids to polyphenols are all significantly above 1. Since flavonoids are a subclass of polyphenols, the concentration of polyphenols should theoretically be greater than the concentration of flavonoids in the same plant. The paper published by Sulaiman 2012, the total phenolic and total flavonoid content was determined in 21 unique plants and each F/P ratio were below 14. Further evidence is provided by Marinova et al in which 42 food products comprised of 20 fruit and 2 vegetable species were tested for polyphenol and flavanoid content. The resultant ratios from the same colorimetric techniques showed F/P ratios significantly below 111. It can be observed that the obtained experimental results are not in agreement with published literature and since the F/P ratio is greater than 1, potentially invalid.

ConclusionThe results obtained from this study for the total phenolic and total flavonoid content of Centipeda Cunninghamii can be used as a basis for further assessment of the preventative nature of this against free radical effects. The flavonoid results and the liquid extract results for the polyphenols will require further assessment to ascertain their proper concentration while the dry herb extract of the polyphenols had provided comparable data to that of HPLC potentially allowing the quicker and faster colorimetric method to be used instead of HPLC.

AcknowledgementsFinancial support from the University of Western Sydney is gratefully acknowledged. We thank Dr. Cheang Khoo for the use of his lab and supervision during the duration of the project. We also thank Mitch Low for providing technical knowledge and support.

References(1) Evans, C. R. (2001): Flavonoid Antioxidants. Current Medicinal Chemistry, 8, 797-807(2) Shahdi, F.; Janitha, P. K.; Wanasundara, P. D. (1992): Phenolic Antioxidants. Critical Reviews in Food Science and Nutrition, 32 (1), 67-103(3) Stevenson, D. E.; Hurst, R. D. (2007): Polyphenolic phytochemicals- just antioxidants or much more?. Cell. Mol. Life Sci, 64, 2900-2916(4) Sulaiman, C. T.; Balachandran, I. (2012): Total Phenolics and Total Flavonoids in Selected Indian Medicinal Plants. Indian J. Pharm. Sci., 74 (3), 258-260(5) Hart, R. S. (2012): That 'Old Man Weed'. Research Directions UWS (6) Beattie, K. D.; Waterman, P. G.; Leach, D. N. (2011): Centipeda cunninghamii, an australian traditional medicinal plant , 12, 77- PL27(7) Evans, C. R.; Miller, N. J.; Bolwell, P. G.; Bramley, P. M.; Pridham, J. B. (1995): The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Rad. Res., 22, 375-383(8) Ainsworth, E. A.; Gillespie, K. M. (2007): Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nature Protocols, 2, 875-877(9) Singleton, V. L.; Rossi Jr, J. A. (1965): Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am. J. Enol. Vitic, 16, 144-158 (10) Zhishen, J.; Mengcheng, T.; Jianming, W. (1999): The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem., 64, 555-559(11) Marinova, D.; Ribarova, F.; Atanassova, M. (2005): Total Phenolics and Total Flavonoids in Bulgarian Fruits and Vegetables. Journal of the University of Chemical Technology and Metallurgy 2005, 40, 255-260

Appendix

Appendix 1: HPLC results for the polyphenols and flavonoids.Hal/Batch NumberSample typePolyphenolic acids mg/gFlavonoids mg/g

201Herbal material12.73.8



204Liquid extract1.700.17

2041.700.17

205Liquid extract1.610.17

1.710.20

determination of total antioxidant content of centipeda cunninghamii

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