Phytochemical and antioxidant properties of some Cassia species

Phytochemical and antioxidant properties of some Cassia species Firdose R. Kolar a *, Chaya L. Gogi a, Mairunisabegum M. Khudavand a, Meera S. Choudhari a and Sindhu B. Patil a a Department of Botany, Katnataka State Women s University, Bijapur 586 109 (Karnataka), India. * Corresponding author: Dr. Firdose R. Kolar Department of Botany Karnataka State Women s University Bijapur 586 109 (Karnataka), India. E-mail: firdose.kousar@gmail.com Telephone : +91-8277284666

Abstract In this study, the antioxidant activity of aqueous and ethanol extracts of four plants from the genus Cassia were evaluated by various antioxidant assays, including ferric reducing antioxidant power (FRAP), DPPH free radical scavenging, metal chelating activity, phosphomolybdenum reducing power, hydrogen peroxide radical scavenging, hydroxyl radical scavenging, deoxyribose degradation and β-carotene bleaching assay. The various antioxidant activities were compared to standard antioxidant such as ascorbic acid. All the extracts showed antioxidant activity in the tested methods. Among the four species Cassia auriculata has been found to possess highest activity in most of the tested models. In addition to the antioxidant activity, the total phenolics and flavonoids were measured in the extracts. The ethanolic extract exhibited highest phenolics and flavonoid contents and had also shown potent antioxidant activity in comparison to the aqueous extracts. The possible antioxidant mechanism of the ethanol extract can be due to its hydrogen or electron donating and direct free radical scavenging properties. Hence, the ethanol extract represents a source of potential antioxidants that could be used in pharmaceutical industries.. Keywords: Cassia, phenolics, flavonoids, free radicals, antioxidants.

Experimental Plant material and preparation of extract The Cassia species namely: Cassia auriculata, Cassia siamea, Cassia uniflora and Cassia italica) were collected from Vijayapur district of Karnataka on 28 th October 2016 and authenticated by Dr. Sidanand Kambhar. The herbaria for voucher specimens of the selected species (C. auriculata: SVK176, C. siamea: SVK177, C. italica: SVK178, C. uniflora: SVK179) were prepared and deposited in the Department of Botany, Karnataka State Women s University, Vijayapur (Karnataka) India. Preparation of extracts The extracts of different plant parts viz. stem, leaves, flowers and pods were prepared using two different solvent systems (distilled water and ethanol). The plant parts were cleaned, dried under shade at room temperature and powdered and about one gram powder of each plant part was treated with 10 ml of distilled water and the extract obtained was shaken vigorously for 5-10 min and left for 24 h in a shaking machine. The obtained extracts were then centrifuged at 10,000 rpm for 15 minutes and the supernatant was collected. The supernatants pooled and the volume was adjusted to 10 ml by dilution with more distilled water. Same procedure was followed for the preparation of ethanol extracts. All the extracts were kept at 4 C and for the assays 1% (v/v) extracts (diluted with double distilled water or respective solvent) were used. Determination of total phenolics and flavonoid content Total phenolic contents (TPC) of the plant extracts were determined using Folin- Ciocalteu method (Wolfe et. al. 2003). The reaction mixture was prepared by mixing an aliquot of the extracts (0.125 ml) with Folin-Ciocalteu reagent (0.125 ml) and 1.25 ml of saturated Na 2 CO 3 solution. Reaction mixture was thereafter incubated for 90min at room temperature. The absorbance was measured at 760 nm. The samples were prepared in triplicates for each analysis and the mean value of absorbance was obtained. A calibration curve was prepared, using a standard solution of Tannic acid (10 μg/ml to 100 μg/ml, R2 = 0.992). Results were expressed in terms of mg tannic acid equivalents (TAE)/ g dry weight (dw) of sample. Total flavonoid contents (TFC) of the plant extracts were analyzed according to the colorimetric method (Luximon-Ramma et al. 2002). The reaction mixture was prepared by adding 1.5 ml of extract to 1.5 ml of 2% methanolic AlCl 3. Samples were incubated for 10min at room temperature. The absorbance was measured at 420 nm. The samples were prepared in

triplicates for each analysis and the mean value of absorbance was obtained. The same procedure was used for the standard solution of rutin and the calibration curve was prepared using a standard solution of rutin (10 μg/ml to 100 μg/ml, R2 = 0.920). The results were expressed on a dry weight (dw) basis as mg rutin equivalents (RE) / g of sample. Ferric reducing antioxidant power assay (FRAP assay) The ability to reduce ferric ions was measured using a method described by Pulido et al. (2000). An aliquot (100μl) of extract was added to 3 ml of FRAP reagent (10 parts of 300 mm sodium acetate buffer at ph 3.6, 1 part of 10 mm TPTZ solution and 1 part of 20 mm FeCl 3. 6H 2 O solution) and the reaction mixture was incubated at 37 C for 15 min. After that, the absorbance was measured at 595 nm. A calibration curve was prepared, using an aqueous solution of ascorbic acid (10 μg/ml to 100 μg/ml, R2 = 0.949). Ascorbic acid is used as positive control and the mixture without the sample is used as the negative control. FRAP values were expressed on a dry weight (dw) basis as milligram of ascorbic acid equivalent per gram of sample. DPPH free radical-scavenging assay The ability of the plant extracts to scavenge DPPH free radical (1,1-diphenyl-2- picrylhydrazyl) was assessed by using the method of Aquino et al. (2001). Plant extract (25µl) was mixed with 3 ml of 25 mm DPPH solution. After 20min incubation in the darkness at room temperature, the absorbance was measured at 515 nm, against a blank of ethanol without DPPH. Results were expressed as percentage of inhibition of the DPPH radical and were calculated by the following formula. Scavenging activity (%) = Absorbance of control - Absorbance of sample Absorbance of control X 100 Phosphomolybdenum reducing power assay The antioxidant activity of the extracts was assessed by the phosphomolybdenum reduction assay according to Prieto et al. (1999). An aliquot of 0.3 ml of sample solution was mixed with 3 ml of the reagent solution (0.6 M sulphuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). The tubes were capped with alluminium foil and incubated at 95 C for 90 min. The tubes were cooled to room temperature and the absorbance of aqueous solution was measured at 695 nm against a blank. The ascorbic acid was used as positive control and a

calibration curve was prepared (10 μg/ml to 100 μg/ml, R2 = 0.984).The reducing capacity of the extracts was expressed as the ascorbic acid equivalents (AAE) per gram of dry weight. Ferrous ion chelating activity The ferrous ion chelating activity was measured by the decrease in the absorbance at 562 nm of iron (II)-ferrozine complex by the method of Dinis et al. (1994). 100 µl of 2 mm Fecl 2 and 300 µl of 5 mm ferrozine were mixed with sample extracts. The mixture was allowed to equilibrate for 10 min before measuring the absorbance. The mixture without the sample is used as positive control and the ability of the sample to chelate ferrous ion was calculated by the formula of inhibition percentage as employed for DPPH free radical scavenging activity. Hydrogen peroxide radical scavenging Hydrogen peroxide radical scavenging activity was measured by replacement titration (Zhao et al. 2006). A solution of 0.2 ml of 40 mm H 2 O 2 and 0.2 ml of test compounds were mixed, followed by 2 drops of 3% ammonium molybdate, 0.2 ml of 2 M H 2 SO 4 and 1.4 ml of 1.8 M KI. The mixed solution was titrated with 5.09 mm NaS 2 O 3 until yellow color disappeared. The relative activities of test compounds to scavenge hydrogen peroxide were expressed as percentage (%) of the titer volume change [(Vcontrol-Vsample)/Vcontrol] X 100. Hydroxyl radical scavenging activity The scavenging activity for hydroxyl radicals was measured with Fenton reaction (Sadasivam and Manikam 1992). Reaction mixture contained 60 μl of 1.0 mm FeCl 3, 90 μl of 1mM 1,10- phenanthroline, 2.4ml of 0.2 M phosphate buffer (ph 7.8), 150 μl of 0.17 M H 2 O 2, and 1.5 ml of extract. Adding H 2 O 2 started the reaction. After incubation at room temperature for 5 min, the absorbance of the mixture at 560nm was measured with a spectrophotometer. The hydroxyl radicals scavenging activity was calculated according to the equation: (A0 A1) / A0 x 100, where A0 is absorbance of the control (without extract) and A1 is the absorbance in the presence of the extract. Deoxyribose degradation assay Deoxyribose is oxidized when exposed to hydroxyl radicals; such degradation can be detected by heating the products in the presence of thiobarbituric acid under acidic conditions, which leads to development of a pink chromogen, by the method of Halliwell et al. (1987), with slight modifications. The assay mixtures, containing the sample, 50 μl of deoxyribose (50 mm), 300μl of phosphate buffer (0.2M, ph 7.4), 50μl of Na 2 EDTA (1 mm), 50μl of FeCl 3 (3.2 mm)

and 50μl of H 2 O 2 (50 mm). The reaction was initiated by mixing 50μl of ascorbic acid (1.8 mm) and the total volume of the reaction mixture was adjusted to 800 μl with buffer. After incubation at 50 o C for 20 min, the reaction was terminated by 250μl of trichloroacetic acid (10%, w/w). The color was then developed by the addition of 150μl of TBA (5%, in 1.25% NaOH aqueous solution) and heating in an oven at 105 o C for 15 min. The mixture was cooled and absorbance was measured at 530 nm against the buffer (as blank). Inhibition of deoxyribose degradation was expressed as: Inhibition% = A0 A A0 X 100 Here, A0 is the A530nm of the mixture without sample solution, and A is A530nm of the reaction mixture with sample solution. β carotene bleaching assay β-carotene bleaching assay was carried out according to the method developed by Wettasinghe & Shahidi (1999). One milliliter of β -carotene solution (0.2 mg/ml chloroform) was pipetted into a round-bottom flask (50ml) containing 0.02 ml of linoleic acid and 0.2 ml of 100% Tween 20. The mixture was then evaporated at 40 C for 10 min using a rotary evaporator to remove chloroform. After evaporation, the mixture was immediately diluted with 100 ml of distilled water. The distilled water was added slowly to the mixture with vigorous agitation to form an emulsion. Three ml aliquots of the emulsion were transferred into different test tubes containing 0.2 ml of samples. The mixture was then gently mixed and placed in a water bath at 50 C for 2h. The absorbance of the sample was measured at 470 nm, immediately after their preparation (t = 0 min) and at incubation times t = 120 min against the blank. Blank solution was prepared, containing the same concentration of emulsion without β-carotene. All determinations were performed in triplicate. The total antioxidant activity was calculated based on the following equation (Miller 1971): % AA = 1- A1 (t=0) A1 (t=120) A0 (t=0) A0 (t=120) X 100 Where:

%AA - Percent of antioxidant activity A1(t=0) - Absorbance of test sample at zero time. A1(t=120) - Absorbance of test sample after 120 min. A0(t=0) - Absorbance of the aqueous control sample at zero time. A0(t = 120) - Absorbance of the aqueous control sample after 120 min. Statistical analysis All the measurements were replicated three times and the results were expressed as mean ± standard deviation. The mean difference among different plant parts was tested by One-way ANOVA and the P<0.05 values were considered significant. Experimental results were further analyzed for Pearson s correlation coefficient of phenolics, flavonoids with different antioxidant assays using MS Excel and GraphPad InStat software. References Aquino R, Morelli S, Lauro MR, Abdo S, Saija A. 2001. Phenolic constituents and antioxidant activity of an extract of Anthurium versicolor leaves. J. Natl. Prod. 64(8): 1019-23. Dinis TCP, Madeira VMC, Almeida MLM. 1994. Free radical scavenging activity of an aqueous extract of potato peel. Food Chem. 85: 611 616. Halliwell B, Gutteridge JMC, Aruoma OI. 1987. The deoxyribose method: a simple test tube assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem. 165: 215-219. Luximon Ramma A, Bahorun T, Soobrattee MA, Aruoma OI. 2002. Antioxidant activities of phenolic, Proanthocyanidin and flavonoid components in extracts of Cassia fistula. J. Agric. Food Chem. 50: 5042-5047. Prieto P, Pineda M, Aguilar M. 1999. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphor molybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 269: 337 341. Pulido R, Bravo L, Saura-Calixto F. 2000. Antioxidant of dietary polyphenols as determined by a modified Ferric Reducing Antioxidant Power assay. J. Agric. Food. Chem. 46: 3396-3402. Sadasivam S, Manikam A. 1992. Biochemical Methods for Agricultural Sciences, New Delhi. Wiley Eastern, 187. Wettasinghe M, Shahidi F. 1999. Antioxidant and Free Radical Scavenging Properties of Ethanolic Extracts of Defatted Borage (Borago officinalis L.) Seeds. Food Chem. 67: 399. Wolfe K, Wu X, Liu RH. 2003. Antioxidant activity of apple peels. J Agric. Food Chem. 51: 609-614.

Zhao GR, Xiang ZJ, Ye TX, Yuan YJ, Guo ZX. 2006. Antioxidant activities of Salvia miltiorrhiza and Panax notoginseng. Food Chem. 99: 767-774.

Table S1. Total phenolics and flavonoid content in different species of Cassia. Species Solvents Total phenolic content (mg TAE/g plant material) Total flavonoid content (mg RE/g plant material) Stem Leaf Flower Pod Stem Leaf Flower Pod Cassia auriculata Cassia italica Cassia siamea Cassia uniflora Aqueous 29.5±0.03 20.7±0.17 32.1±0.53 9.12±0.03 0.65±0.02 1.35±0.01 2.38±0.05 1.13±0.02 Ethanol 51.1±0.27 76.2±0.12 80.4±1.78 24.3±0.01 1.32±0.02 3.07±0.02 4.32±0.02 2.77±0.10 Aqueous 4.55±0.02 10.3±0.01 27.7±0.01 7.38±0.02 0.45±0.02 1.53±0.04 2.86±0.02 0.47±0.07 Ethanol 5.78±0.01 15.8±0.03 39.5±0.17 8.74±0.02 1.01±0.02 3.79±0.02 4.00±0.01 1.51±0.01 Aqueous 9.21±0.01 27.7±0.02 11.3±0.01 32.6±0.27 0.65±0.02 1.28±0.02 0.98±0.01 1.42±0.01 Ethanol 22.9±0.04 39.1±0.27 30.1±0.02 45.7±0.17 0.96±0.02 1.92±0.02 1.69±0.01 2.22±0.01 Aqueous 6.08±0.02 12.5±0.03 38.5±0.01 12.6±0.01 0.26±0.02 0.36±0.01 1.36±0.02 0.51±0.02 Ethanol 11.5±0.03 20.6±0.02 57.7±0.02 15.1±0.01 1.22±0.02 3.79±0.02 3.97±0.02 1.74±0.13 Values are expressed as mean ± SD of triplicate measurements. mg TAE/ g plant material: milligram tannic acid equivalent per gram plant material. mg RE/g plant material: milligram rutin equivalent per gram plant material.

Table S2: Antioxidant activity in different species of Cassia. Species Solvents Ferric reducing antioxidant power ( mg AAE /g plant material) DPPH free radical scavenging activity (%) Stem Leaf Flower Pod Stem Leaf Flower Pod Cassia auriculata Cassia italica Cassia siamea Cassia uniflora Aqueous 42.18±0.03 109.6±0.03 142.7±0.05 35.88±0.05 81.42 78.95 85.56 72.85 Ethanol 79.44±0.59 161.1±0.39 161.5±0.20 43.57±0.05 85.82 82.03 86.09 75.15 Aqueous 11.56±0.03 19.71±1.33 53.44±0.05 14.51±0.05 43.79 63.02 67.14 62.36 Ethanol 18.16±0.02 31.03±0.02 73.34±0.22 15.05±0.05 67.85 68.46 72.47 66.70 Aqueous 34.21±0.05 79.21±0.39 57.73±0.05 79.89±0.39 67.30 64.45 70.65 65.54 Ethanol 49.58±0.05 93.43±0.09 67.52±0.05 109.9±0.59 69.56 69.94 85.87 73.18 Aqueous 18.40±0.02 38.12±0.03 105.3±0.03 15.12±0.05 60.54 55.65 76.09 57.08 Ethanol 30.03±0.05 51.65±0.03 126.5±0.22 30.26±7.48 64.39 61.15 80.49 61.20 Values are expressed as mean ± SD of triplicate measurements. mg AAE /g : milli gram ascorbic acid equivalent per gram plant material.

Table S3: Antioxidant activity in different species of Cassia. Species Solvents Phosphomolybdenum reducing power ( mg AAE /g plant material) Ferrous ion chelating Activity (%) Stem Leaf Flower Pod Stem Leaf Flower Pod Cassia auriculata Cassia italica Cassia siamea Cassia uniflora Aqueous 27.5±0.14 41.23±0.08 44.7±0.40 8.15±0.37 51.22 67.84 87.72 42.37 Ethanol 38.6±0.18 52.9±0.28 63.8±0.37 12.5±0.02 70.98 80.51 90.05 69.20 Aqueous 8.09±0.02 22.3±0.21 40.6±0.47 19.4±0.07 76.54 82.77 79.91 73.43 Ethanol 12.06±0.02 29.8±0.02 60.7±0.32 25.2±0.03 79.62 83.10 89.50 82.69 Aqueous 8.92±0.04 38.8±0.47 15.9±0.37 43.4±0.47 74.82 78.50 80.49 84.23 Ethanol 20.09±0.02 41.5±0.02 35.2±0.03 47.8±0.03 80.89 89.75 82.56 87.12 Aqueous 10.4±0.19 19.3±0.11 27.6±0.28 16.5±0.21 58.44 66.71 73.77 62.12 Ethanol 13.38±0.04 25.4±0.02 41.8±0.39 24.3±0.02 69.48 71.87 81.28 79.91 Values are expressed as mean ± SD of triplicate measurements. mg AAE /g : milli gram ascorbic acid equivalent per gram plant material

Table S4: Antioxidant activity in different species of Cassia. Species Cassia auriculata Cassia italica Cassia siamea Cassia uniflora Solvents Hydrogen Peroxide radical scavenging activity (%) Hydroxyl Radical Scavenging Activity (%) Stem Leaf Flower Pod Stem Leaf Flower Pod Aqueous 20.21 26.66 30.34 26.66 42.62 59.40 72.36 44.75 Ethanol 26.66 36.66 53.23 46.66 48.83 64.05 89.28 54.74 Aqueous 21.08 23.33 26.66 20.29 33.43 76.14 85.01 59.25 Ethanol 30.20 32.66 43.11 34.14 41.86 78.42 87.45 65.72 Aqueous 24.09 26.14 20.98 36.66 40.04 57.39 60.14 71.74 Ethanol 26.66 36.61 33.33 40.08 60.73 74.62 73.06 88.25 Aqueous 16.66 19.66 24.53 26.66 33.87 50.97 54.17 36.92 Ethanol 30.21 30.87 23.33 39.17 58.39 57.53 66.43 52.87 Values are expressed as mean ± SD of triplicate measurements.

Table S5: Antioxidant activity in different species of Cassia. Species Solvents Deoxyribose degradation Activity (%) β- Carotene bleaching activity (%) Stem Leaf Flower Pod Stem Leaf Flower Pod Cassia auriculata Cassia italica Cassia siamea Cassia uniflora Aqueous 77.16 74.02 80.28 59.53 52.65 34.27 68.19 36.39 Ethanol 81.94 86.35 89.57 71.15 67.47 78.09 86.46 45.15 Aqueous 75.83 78.37 83.51 75.37 70.67 67.84 32.15 83.74 Ethanol 79.16 82.73 85.64 81.48 75.75 79.85 44.52 85.86 Aqueous 63.33 67.91 73.98 74.44 16.60 28.62 39.57 53.17 Ethanol 72.18 77.50 85.78 78.70 42.40 64.66 57.38 76.92 Aqueous 62.17 67.03 76.03 58.28 45.58 74.91 78.44 73.14 Ethanol 66.99 77.29 78.14 61.42 49.85 75.75 84.34 77.69 Values are expressed as mean ± SD of triplicate measurements.

Table S6: ANOVA for antioxidant activity among different part extracts by different antioxidant assays from four Cassia species. Antioxidant assays df MS P FRAP 3 6713 0.008 DPPH 3 227.2 0.068 MoO 2 P reduction 3 876.1 0.006 Fe2+ chelation 3 262.1 0.083 H 2 O 2 scavenging 3 131.7 0.132 OH scavenging 3 1146 0.001 Deoxyribose degradation 3 206.0 0.019 β- Carotene bleaching 3 278.9 0.554

Table S7: Regression coefficients (R2 value) for antioxidant activity assessed by different methods and the relative influence of antioxidant components. Species FRAP DPPH Cassia auriculata Cassia italica Cassia siamea Cassia uniflora TPC TFC TPC TFC TPC TFC TPC TFC Data were statistically analyzed using Pearson correlation coefficient test. **Indicates a significant difference at the level of p < 0.01 *Indicates a significant difference at the level of p < 0.05 ns Indicates not significant at the level of p>0.05 MoO 2 P reduction Fe2+ chelation H 2 O 2 scavenging OH scavenging Deoxyribose degradation β- Carotene bleaching Aqueous 0.287 ns 0.950* 0.586 ns 0.463 ns 0.004 ns 0.234 ns 0.946* 0.935 * Ethanol 0.961* 0.559 ns 0.976* 0.779 ns 0.013 ns 0.751 ns 0.967* 0.972* Aqueous 0.775 ns 0.296 ns 0.356 ns 0.768 ns 0.826 ns 0.899 ns 0.142 ns 0.336 ns Ethanol 0.399 ns 0.001 ns 0.263 ns 0.698 ns 0.802 ns 0.863 ns 0.192 ns 0.233 ns Aqueous 0.996** 0.452 ns 0.934* 0.178 ns 0.888 ns 0.624 ns 0.939* 0.879 ns Ethanol 0.984** 0.928* 0.965* 0.948* 0.909* 0.658 ns 0.989** 0.856 ns Aqueous 0.912* 0.445 ns 0.880 ns 0.430 ns 0.985** 0.714 ns 0.985** 0.880 ns Ethanol 0.618 ns 0.544 ns 0.650 ns 0.614 ns 0.436 ns 0.841 ns 0.790 ns 0.348 ns Aqueous 0.837 ns 0.585 ns 0.987** 0.463 ns 0.695 ns 0.561 ns 0.172 ns 0.384 ns Ethanol 0.999** 0.015 ns 0.939* 0.721 ns 0.961* 0.895 ns 0.053 ns 0.970* Aqueous 0.973* 0.291 ns 0.941* 0.680 ns 0.507 ns 0.807 ns 0.429 ns 0.298 ns Ethanol 0.906* 0.022 ns 0.997** 0.629 ns 0.991** 0.903* 0.247 ns 0.967* Aqueous 0.935* 0.826 ns 0.883 ns 0.863 ns 0.960* 0.573 ns 0.755 ns 0.405 ns Ethanol 0.991** 0.898 ns 0.891 ns 0.420 ns 0.595 ns 0.926* 0.466 ns 0.427 ns Aqueous 0.879 ns 0.861 ns 0.804 ns 0.767 ns 0.322 ns 0.445 ns 0.661 ns 0.347 ns Ethanol 0.592 ns 0.268 ns 0.655 ns 0.107 ns 0.367 ns 0.379 ns 0.822 ns 0.543 ns