Ultrasound-Assisted Extraction in Different Solvents for Phytochemical Study of Canna indica

International Journal of FoodEngineering

Volume 6, Issue 3 2010 Article 6

Ultrasound-Assisted Extraction in DifferentSolvents for Phytochemical Study of Canna

indica

Padma S. Vankar∗ Jyoti Srivastava†

∗Indian Institute of Technology, [email protected]†Indian Institute of Technology, [email protected]

Copyright c©2010 The Berkeley Electronic Press. All rights reserved.

Ultrasound-Assisted Extraction in DifferentSolvents for Phytochemical Study of Canna

indica

Padma S. Vankar and Jyoti Srivastava

Abstract

Red flowers of Canna indica (Family Cannacece) were extracted in Sonicator with differentsolvents for the estimation of polyphenols and flavonoids using fresh as well as dry flowers. An-tioxidant capacities of L- Ascorbic acid, trolox, pyrogallol and Canna flower extracts were alsoevaluated based on its ability to scavenge the DPPH radical, results obtained were compared withthose of TEAC aay. A good correlation was observed between the two methods. The resultsshowed that acetone extract of fresh flowers contained the highest amount of antioxidant activity78.33 %, 89.7 mg GAE / g and 51.45 µMT / g although the yield of the extract in the methanolwas highest (34 g / 100 g). Total phenol and flavonoids content (0.96 mg GAE / 100 g) (19.89 mgQE / 100 g) were also evaluated for these extract. This is the first report on the phytochemicalsand antioxidant activity from Canna indica flowers particularly for its radical scavenging activityand can be a good source of food additives.

KEYWORDS: Canna indica, polyphenols, flavonoids, antioxidant activity, radical scavenging

1. INTRODUCTION In recent years research interest has been growing widely to study the detailed phytochemicals in medicinal plants. The plant based phytochemicals are classified into various classes such as polyphenols, flavonoids, carotenoids, alkaloids, tannin etc. Flavonoids are a class of low molecular weight, naturally occurring polyphenolic compounds widely distributed in the plant kingdom. They exhibit different biological functions that allow interactions between plants and their environment. The phenolics present in flavonoids are also potent antioxidants when tested in vitro [1]. Flavonoids like many other polyphenols are excellent free radical scavengers (chain-breaking antioxidants) because they are highly reactive as hydrogen or electron donors [2-6]. Flavonoids have been known to offer protection against major diseases such as coronary heart diseases and cancer [7] and they also exhibit biological activities, including antiallergenic, antiviral, anti-inflammatory, antioxidant, antithrombotic, antiviral and anticarcinogenic activities. However, most interest has been devoted to their antioxidant activity, which is due to their ability to reduce free radical formation and also to scavenge free radicals [8-10].

Today's toxic environment and modern life styles generate stress which has enormous impact on our health, this increased stress culminates in formation of large amount of free-radicals. These free radicals must be balanced or deactivated to maintain good health. Due to biochemical processes, it is normal for free radicals to be present in the body at all times. A normal healthy immune system is normally able to control the existence of free radicals and minimize their potential damage. Free radicals and other reactive oxygen species are considered to be important causative factors in the development of diseases such as neurodegenerative diseases cancer and cardiovascular diseases.

Interest in the role of antioxidants in human health has prompted research in the fields of horticulture and food science to assess fruit and vegetable for their antioxidant contents, such as ascorbate, carotenoids, tocopherols, and phenolics. Natural antioxidants such as α-tocopherol and L-ascorbic acid are widely used because they are seen as being safer and causing fewer side reactions, but their antioxidant activities are, however, lower than those of synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene, tertiary butylated hydroquinone and gallic acid esters. But synthetic antioxidants have been suspected to cause or prompt negative health effects too. Hence, strong restrictions have been placed on their application and there is a trend to substitute them with naturally occurring antioxidants. Moreover, these synthetic antioxidants also show low solubility [11].

Hence, there is a need for safe and economic viable sources of antioxidants which have high activity from natural sources to replace these

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Vankar and Srivastava: Phytochemical Study of Canna

Published by The Berkeley Electronic Press, 2010

synthetic antioxidants. The antioxidant compounds present in edible plants have recently been promoted as food additives because they display little or no toxic side effects.

Therefore, there is a growing interest in substances exhibiting antioxidant properties that are supplied to human and animal organisms as food components or as specific preventive pharmaceuticals. The plant kingdom offers a wide range of natural antioxidants. However, there is still not enough knowledge about the practical usefulness of most of them. In the group of secondary plant metabolites, antioxidant phenolics are commonly found in various fruits, vegetables and herbs and they have been shown to provide a defence against oxidative stress from oxidizing agents and free radicals

Canna indica is a wildflower, commonly called "Indian shot" and it is widely cultivated in India belongs to the family Cannaceae. Rhizome of Canna indica has been used in traditional folk medicines. In the process of screening of some plants, interest in red Canna flower (local name-Keli) led to this study. The bright red color of this flower makes it a valuable ornamental plant and a potential source for extraction of natural colorants for dyeing different types of textiles [12].

In this study we have investigated the antioxidant activity in commonly found red flower in India. We have compared Antioxidant activity in the flower in different extracts paying special attention to this flower not previously analysed.

The aim of this paper was to evaluate the antioxidant properties of the different extracts from Canna indica (crude methanol, ethyl acetate, acetone, ethanol and water) and to correlate their antioxidant potential to the composition of polyphenols. 2. MATERIALS AND METHODS 2.1 Chemical and spectral measurements: All the solvents used for extraction were AR grade and for quantitative analysis HPLC grade were obtained from S.D Fine chemicals- Gallic acid, Sodium carbonate, Folin-Ciocalteu (2N), Aluminium chloride, Potassium persulfate, Sodium nitrite, Pyrogallol, Phosphate buffer, Potassium ferricyanide, Trichloroacetic acid, Ferric chloride, DPPH (Alfa aser 95%), L- Ascorbic acid (Aldrich), Quercetin-98% (Hi Media), ABTS (Fluka), Trolox (Aldrich), Distilled water (Millipore). 2.2 Plant Material: The red flowers of Canna indica was collected from I.I.T. Campus, Kanpur, India and was identified at the C. S.A. Agricultural University, Kanpur.

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International Journal of Food Engineering, Vol. 6 [2010], Iss. 3, Art. 6

http://www.bepress.com/ijfe/vol6/iss3/art6DOI: 10.2202/1556-3758.1599

2.3.1 Conventional extraction: Extraction with organic solvents methanol, acetone, ethyl acetate, ethanol were done in a soxhlet apparatus (borosil) for 10-12 hrs and aqueous extraction is done on water bath in a beaker. Canna indica fresh and dry petals were extracted in different solvents and water (100g in 400 ml of solvent each) at 600C temperature, the extracted material was recovered by vacuo filtration through Whatman- 42 paper (Sortorius) in a Buchner funnel. In all cases, the solvent was evaporated in a rotavapor (Buchi R-210) at 650C to near dryness, and then dried in high vacuo for 24 h to achieve complete dryness and the extract yield was determined as the increased weight of the flasks. 2.3.2 Ultra sonication-Assisted extraction: Each 20 g of flower was mixed with 50 ml of extracting solvents (Methanol, Ethanol, Ethyl-acetate, Acetone and water) in a 100 ml conical (Tarsons) 4.0 cm in diameter. The ultrasound chamber (JULABO, USR3) having a frequency of 20 using different sonic power (100–500 W) for different time intervals (15–120 min). The power level was set at the maximum (level 9) and the temperature during the 1–2 hour extraction period was stabilized at 25-30 0C during the extraction. For indirect sonication, the sample (conical) was immersed in an ultrasound chamber at the half height of the solution in the flask, operating at 20 kHz frequency. The temperature was controlled and maintained at below 30 0C by periodical replacing water in the bath with cold one (15 0C). The flask was taken out and cooled to room temperature by cooling water. The flower extracts were filtered through Whatman No. 42 paper and the solution was collected. The residue was taken back and extracted again in the same conditions. The extracts of the twice-extraction were mixed. The ultrasound power actually delivered to the extracting liquid by the sonic bath was determined by the calorimetric method. This suggests that the Ultra sonication-Assisted extraction process is more selective than the conventional one. 2.4 Total Phenolic Content: The total phenolic content (TPC) in the extract of different solvents with dry and fresh flowers was determined using Folin –Ciocalteu reagent [13]. To the 0.1 g of the dry extract 4.5 ml of de-ionized water, 0.5 ml filtrate, 0.2ml of 2N Folin-Ciocalteu reagent, 0.5ml of 2.5% sodium carbonate and finally 4.3 ml of de-ionized water were added and mixed completely. After 2 hours, the absorbance of the solution at 725 nm was measured in triplicate in a spectrophotometer (Heλios α, Thermo Electron Corp.). Quantification was based on the standard curve of Gallic acid (0-0.5 mg/ml), which was dissolved in de-ionized water and expressed as Gallic Acid Equivalent per 100 g extract. 2.5 Total Flavonoids Content: Total Flavonoids Content (TFC) of different solvent extracts of Canna indica flowers was determined by Zhishen et al.,[14]. A

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0.5 ml aliquot of the flower extract, (10mg/10ml), was mixed with 0.4 ml distilled water, 0.15 ml of 5 % sodium nitrite was added and the mixture was allowed to react for 5 min. Following this 0.15 ml of 10% Aluminium Chloride was added and the mixture was made to stand for 5 min further. Finally, the reaction mixture was diluted with distilled water and the absorbance at 510 nm was recorded in triplicate against a blank. Total flavonoid content was calculated using Quercetin as standard and expressed as mg Quercetin equivalents. 2.6 Determination of Antioxidant activity: Different solvent extracts of Canna indica flowers often exhibit a strong antioxidant activity, notably due to the presence of hydroxyl groups and aromatic heterocyclic rings that provide these molecules with hydrophilicity and particular stability. 2.6.1 DPPH Assay: Antioxidant activity (AOA) assay was done by the DPPH method described by Sanchez-Moreno (1990) [15]. The different extracts were measured in terms of hydrogen donating or radical scavenging ability using a stable radical DPPH. 2.8 ml of DPPH solution (0.045 mg/ ml) were rapidly mixed with 0.2 ml and 0.4 ml of methanolic solution of plant extract (0.0025 g/10 ml) one at a time in cuvette placed in the spectrophotometer. The absorbance at 515 nm was measured after 5 min. The initial absorbance of the DPPH was 1.2 -1.3. The decline in radical concentration indicated the radical scavenging activity of the sample. Pyrogallol was used as reference corresponding to 100% radical scavenging activity. Radical scavenging activity was evaluated as percentage was calculated as shown in equation (1):

(A0 - Atest) / (A0 - Aref ) X 100 Eq (1) where A0 is the initial absorbance (DPPH + Sample) and Aref and Atest are absorbance after 5 min with pyrogallol solution and sample solution. All measurements were done in triplicate. 2.6.2 Trolox equivalent antioxidant capacity (TEAC) assay: Antioxidant capacity of different extracts were evaluated by the improved ABTS *+ (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical method as described by Re et al., (1999) [16] with slight modification. ABTS *+ radical cation was generated by a reaction of 7 mM ABTS with 2.45 mM potassium persulphate. The reaction mixture was allowed to stand in the dark for 16 hrs at room temperature and used within two days. The ABTS*+ solution was diluted with deionized water to give an absorbance of 0.700 at 734 nm. All samples were diluted appropriately to give absorbance values 20-80% of the blank. Trolox

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solution was used as reference standard. The ABTS*+ scavenging effect (%) is calculated as shown in equation (2):

(OD 734 standard - OD 734 sample)/ OD 734 standard) X 100 . Eq (2) 2.4.3 Evaluation of Antioxidant Activity in FRAP system: The Ferric Reducing Antioxidant Power of Canna indica was evaluated according to Oyaizu, (1986) [17]. Different extracts were suspended in distilled water and mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6), and 2.5 ml of 1% Potassium ferricyanide. The mixture was incubated at 323 K for 20 min, 2.5 ml of 10 % Trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and Ferric chloride (0.5 ml, 0.1 %), and the absorbance was measured at 700 nm. Increase in absorbance of the reaction mixture indicated reducing power. The standard curve was prepared using 2, 4, 6, 8, 10 mg l-1 solutions of Gallic acid in methanol: water (50:50, v/v). Total phenol values were expressed in terms of Gallic acid equivalent (mg g –1 of dry mass). 2.5 Statistical analyses: All the experiments were carried in triplicates and the experimental results represent treatment groups expressed as means ± SD. 3. RESULT AND DISCUSSION Canna indica flowers have been extracted with different solvents and then various phytochemical analyses have been carried out with these extracts. The presence of high content of polyphenols and flavonoids has been shown to be the cause of good antioxidant activity in these extracts. 3.1 Optimization of ultrasonic extraction: In this work, a new extracting method involving ultrasonic treatment was proposed. Previous studies have shown that the Conventional extraction (Soxhlet extraction) of pigment in different solvents is temperature dependant. The results of the Ultrasound Assisted Extraction experiments indicated that ultrasound enhanced the efficiency of Canna flower extraction by shortening the extract time and lowering the volume of the solvents needed, but at the same time. This method did not increase the yield very much on comparing with conventional solvent extraction. The reason of this phenomenon might be due to the fact that the hydroxyl radicals produced by acoustic cavitation of ultrasound in extracts, with the presentation of a small amount of water in the fresh red Canna flowers. To fully investigate the effect of the Ultrasonic efficiency of the extraction, the temperature of the extraction mixture was reduced below its boiling point. This also produced a

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pronounced increase in the color strength of the extract than the Conventional extraction method. It can be observed from Fig 1 that Ultrasound Assisted Extraction is better option in comparison with Conventional extractability. 3.2 Determination of yield: This is a first study on evaluation of phytochemicals in red Canna indica flower which showed that among the various solvent- methanol, acetone, ethanol, ethyl acetate and aqueous extracts of Canna indica. The extraction yields for fresh and dry petals of red Canna, obtained employing the selected solvents are shown in Fig 1. Ultrasonic Methanolic extract of fresh Canna (MFC) flower showed highest yield 34.09 g/100g whereas as conventional method showed yield 30.19 g/100g. Similarly, ultrasonic Ethyl acetate extract of dry Canna (EADC) flower showed the lowest yield 7.24 g/100g whereas conventional method showed 3.99 g/100g. Among all the solvents used in this study, acetone extract of fresh flowers yielded second highest amount of crude extract (20.43g/100g) which showed good and highest level of polyphenolics TPC, TFC and AOA. This study demonstrates that these disadvantages of using Soxhlet extraction method could be overcome by using ultrasound assisted solvent extraction. 3.3 Determination of phenolic compounds in red Canna flowers: The phenol concentrations in these organic solvent extracts and water extracts were found to be high. It was determined from regression equation of calibration curve (y = 10.738x + 0.061, R 2= 0.98). The values of TPC varied between from 0.06 to 0.96 mg Gallic acid equivalent per 100 g of different solvent extracts as shown in table 1. The levels of total phenols determined in this way are not absolute measurements of the amounts of phenolic materials but are in fact based on their chemical reducing capacity relative to an equivalent reducing capacity of Gallic acid. The different responses of the extracts in this assay may arise from the solvent and variety of phenolics found in Canna. The Acetone extracts of both dry (ACDC) and fresh (ACFC) had higher total phenolic compound (0.96 mg GAE/g) as compared to other solvents and aqueous extracts. 3.4 Determination of flavonoids in red Canna flower: Flavonoids content were expressed in (mg/g), in Quercetin equivalents. The samples showed values which varied from 1.76 to 19.89. The highest amounts of flavonoids were found in acetone extract of fresh (ACFC) flower of Canna, which contained the highest amount of phenolics as well. It was observed that the amount of flavonoids showed direct correlation with the total amount of phenolics. The high flavonoid content in acetone extract of red Canna (ACFC) contributes largely to its increased antioxidant potential in comparison to other extracts. Table 2 shows the

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values of total phenolic contents and total flavonoid contents in ten different extracts. 3.5 Determination of antioxidant activities in red Canna flowers: The acetone fresh flower extract possesses significantly high antioxidant activity and is most potent extract which showed good free radical scavenging effect as assessed by the DPPH, TEAC and FRAP assays whereas the ethanolic dry flower extract showed the weakest antioxidant activity. Different solvent extracts showed different antioxidant activity due to the difference in concentrations of anthocyanin, total phenols and flavonoids which got extracted from the dry and fresh flowers of Canna indica. Hence, two methods have been used to measure their antioxidant activities. Furthermore, these methods are reliable and easy to perform and can be used for determining hydrophilic and lipophilic antioxidant capabilities of samples. Results suggest that the red fresh flowers of Canna indica could be a potential candidate for natural antioxidant 3.5.1 Total antioxidant capacity of Canna flower by ABTS *+ decolorization assay: This method measures the relative antioxidant ability of flower to scavenge the radical ABTS *+ the aqueous phase, as compared with a standard amount of Trolox. The ABTS *+, generated by potassium persulfate, is presented as an excellent tool for determining the antioxidant activity of hydrogen-donating antioxidants (scavengers of aqueous phase radicals) and of chain breaking antioxidants (scavengers of lipid peroxyl radicals). The antioxidant defense system of the body is composed of a mixture of antioxidants. ABTS decolorization assay has been used for the first time to evaluate the antioxidant capacity of different extracts of Canna indica flower. As shown in Table 2, total antioxidant capacity, in TEAC of flower tested, was found to range from 6.67-51.45 μMT/g. The activity of the acetone (ACFC) flower extract was found to show highest free radical scavenging effect while the ethanolic extract of dry flowers (EDC) showed the lowest antioxidant activity. The results demonstrate that Canna indica extract possesses an interesting antioxidant activity and is a potential candidate for natural antioxidant. 3.5.2 Antioxidant activity by DPPH free radicals: All the extracts were studied for antioxidant properties by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as well. The efficiency of each extract differed depending on the particular solvent type and methodology involved in total antioxidant capacity. The study revealed that the extracts showed good antioxidant activity by this assay. The AOA of extract of acetone (ACFC) showed the highest inhibition towards DPPH radicals (78.33%) with respect to the control value by 50% (IC50). These results demonstrated similar trend as the ABTS assay in antioxidant activity.

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3.5.3 Determination of Reducing power by FRAP Assay: There is a wide range of antioxidant activities within the extracts analyzed, as measured by the FRAP (ferric reducing ability of plasma) method. The values of absorbance at 700 nm for the ten crude extracts in different solvents of Canna flowers revealed that all samples had a capacity to reduce iron (III), and the Reducing Power (RP) values of the samples were significantly different, ranging from the lowest value of 11.9 (EDC) to the highest value of 89.7 mg GAE/g (ACFC). Based on the antioxidant data obtained, Extract with FRAP antioxidant capacity high values will be further investigated in the future to determine which chemical groups are responsible for such high antioxidant capacity values. 3.6 Correlation of ABTS assay and DPPH assay: L- Ascorbic acid (AA) is one of the most effective antioxidants in fruits and vegetables. People with high intakes of dietary AA acid or citrus fruits have repeatedly been associated with lowered risk of developing cancer. The AA contribution to scavenge ABTS *+ varies extensively up to 200 times the lowest value. A linear correlation between reduced ABTS *+ and DPPH after different concentrations of AA were added. Similar results were obtained when Trolox and Pyrogallol were added, and their linear correlation could be described as [ABTS *+] = 0.963[DPPH] (R2 = 0.9993), [ABTS *+] = 0.9987 [DPPH] (R2 = 0.9989) and [ABTS *+] = 1.688 [DPPH] (R2 = 0.9989) respectively. In this study, it is found that 1 mol of AA reacts with approximately 2 mols of ABTS *+ radicals or DPPH· radicals. 1 mol of AA reduces 2 mols of ABTS *+ radicals similarly, 2 mols of ABTS *+ or DPPH·radicals are scavenged by 1 mol of trolox. The stoichiometry of the reaction between pyrogallol and ABTS *+ or DPPH· radical is found to be 1:5 and 1:4, respectively. For the antioxidants tested, the stoichiometry of reactions between antioxidant and ABTS *+ or DPPH· is very close. However, when pyrogallol was used, the stoichiometry differs. The total antioxidant potential of Canna flowers showed significant similarity by employing TEAC and DPPH scavenging assay. However, the results obtained in this study, show a good correlation between the two models. 4. Conclusions The Ultrasound Assisted Extraction method reported here can offer an effective alternative for simultaneous extraction of pigment (phytochemicals) from Canna indica. The optimization process involved studying the response of the experimental conditions is best and it is proved to be an effective method for the extraction. Results obtained indicated that all the extracts from Canna indica have a great potential as a source of natural antioxidant agent, which contain fairly high amount of flavonoid and phenolic compounds, which is exhibited

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through good antioxidant activity. The high scavenging property of flower extracts may be due presence of large contents of phenolic compounds, the hydroxyl groups present in the molecules can provide the necessary component as a radical scavenger. Table 1 Total Phenolic Contents and Total Flavonoid Contents in different extracts

Extract TPC(mg GAE/100g) TFC(mg QE/100g)

MFC 0.54 ± 0.15 8.95 ± 0.09 MDC 0.15 ± 0.03 2.18 ± 0.04 AFC 0.34 ± 0.08 4.58 ± 0.06 ADC 0.10 ± 0.03 2.01 ± 0.04 EAFC 0.78 ± 0.17 12.44 ± 1.45 EADC 0.29 ± 0.05 6.89 ± 0.06 ACFC 0.96 ± 0.19 19.89 ± 1.79 ACDC 0.37 ± 0.11 9.85 ± 1.00 EFC 0.31 ± 0.06 4.00 ± 0.05 EDC 0.06 ± 0.01 1.76 ± 0.02 *MFC-Methanolic fresh canna and MDC-Methanolic dry canna AFC-Aqueous fresh canna and ADC-Aqueous dry canna EAFC-Ethyl acetate fresh canna and EADC-Ethyl acetate dry canna ACFC-Acetone fresh canna and ACDC-Acetone dry canna EFC-Ethanolic fresh canna EDC-Ethanolic dry canna

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Table 2 Radical Scavenging Capacity of different samples of Canna indica by three different methods

Extract DPPH(%Inhibition) FRAP(mgGAE/g) TEACµMT/gMFC 37.00 66.6 ± 2.1 14.96 ± 0.15

MDC 11.80 35.3 ± 0.7 11.54 ±1.45

AFC 18.70 58.5 ± 1.5 12.02 ±0.51

ADC 7.00 21.2 ± 0.5 8.56 ± 0.23

EAFC 54.09 70.7 ± 2.7 20.22 ± 0.15

EADC 27.66 43.4 ± 0.8 16.78 ±1.45

ACFC 78.33 89.7 ± 2.9 51.45 ± 0.98

ACDC 44.68 62.6 ± 1.7 43.56 ± 0.67

EFC 16.98 51.5 ± 0.8 10.18 ±1.78

EDC 5.78 11.9 ± 0.5 6.67±1.45

*C-Ex- Conventional extraction; U-Ex- Ultrasonic extraction

Figure 1: Percentage Yield in different solvent Ultrasonic assisted extracts

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REFERENCES [1] C. A. Rice-Evans, N.J. Miller, P. G. Bolwell, P.M. Bramley, J.B. Pridham.

Free Radical Research, 22 (1995) 375–383. [2] N. Cotelle. Curr. Top. Med. Chem., 1 (2001) 569–590. [3] C. Kaur and H. Kapoor. Int. J. Food Sci. Technol., 36 (2001) 703–725. [4] A. S. Pannala, T. S. Chan, P. J. O’Brien, C. A. Rice-Evans. Biochem.

Biophys. Res. Commun., 282 (2001) 1161–1168. [5] B. Yang, A. Kotani, K. Arai, F. Kusu. Anal. Sci., (2001), 599–604. [6] O. Blokhina, E. Virolainen, K.V. Fagerstedt. Ann. Bot., 91 (2003) 179–194. [7] M. G. Hertog and P. C. Hollman. Eur J Clin Nutr., 50 (2) (1996) 63-71. [8] A. L. Miller. Alt. Med. Rev., 1 (1996) 103–111. [9] G. Mojzˇisˇova´ and M. Kuchta. Physiol. Res., 50 (2001) 529–535. [10] P. K. Knekt, J. Kumpulainen, R. Jarvinen, H. Rissanen, M. Heliovaara, A.

Reunanen, T. Hakulinen, A. Aroma. Am. J. Clin. Nutr., 76 (2002) 560–568.

[11] A. L. Branen, J. American Oil Chemists Society, 5 (1975) 59- 63. [12] B. Ghorpade, V. Tiwari, P. S. Vankar. Asian Textile Journal, (2000) 68-69. [13] M. S. Taga, E. E. Miller, D. E. Pratt, J Am Oil Chem Soc., 61 (1984) 928-

931. [14] J. Zhishen, T. Mengcheng, W. Jianming. Food Chemistry, 64(4) (1999) 555–

559. [15] C .Sanchez-Moreno. Food Science and Technology International, 8(3) (1990)

21–137. [16] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M.Yang, C. A. Rice-Evans.

Free Radical Biology and Medicine, 26 (1999) 1231–1237. [17] M. Oyaizu. Japanese Journal of Nutrition, 44 (1986) 307–315.

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Ultrasound-Assisted Extraction in Different Solvents for Phytochemical Study of Canna indica

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