Research Article Received: 6 June 2014
Revised: 13 August 2014
Accepted article published: 20 August 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6879
Effect of different drying methods on chlorophyll, ascorbic acid and antioxidant compounds retention of leaves of Hibiscus sabdariffa L. Sandopu Sravan Kumar, Prabhakaran Manoj, Nandini P Shetty and Parvatam Giridhar* Abstract BACKGROUND: Use of the indigenous, easily accessible leafy vegetable roselle (Hibiscus sabdariffa L.) for value addition is gaining impetus as its nutritive and nutraceutical compounds are exposed by investigations. Being a perishable, storage is challenging, hence different methods of drying have been an attractive alternative for its postharvest usage in foods without much compromising its quality and antioxidant potential. RESULTS: Room- and freeze-dried samples were found to have best quality in terms of colour, total flavonoid content (18.53 ± 2.39 and 18.66 ± 1.06 g kg−1 respectively), total phenolic content (17.76 ± 1.93 and 18.91 ± 0.48 g kg−1 ), chlorophyll content (1.59 ± 0.001 and 1.55 ± 0.001 g kg−1 ) and ascorbic acid content (11.11 ± 1.04 and 8.92 ± 0.94 g kg−1 ) compared with those subjected to infrared, crossflow, microwave, oven or sun drying. Samples treated by room and freeze drying retained maximum antioxidant potential as shown by the phosphomolybdate method and the 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity and ferric-reducing antioxidant power assays. Cold water and hot water extracts showed significantly higher total phenolic content and total antioxidant activity owing to the greater solubility of phenolics and destruction of cellular components in polar solvents than in organic solvents. CONCLUSION: The data obtained show the potential for retaining quality parameters of roselle leaf under suitable drying methods. © 2014 Society of Chemical Industry Keywords: roselle; drying methods; ascorbic acid; antioxidant properties; sun drying; microwave drying
INTRODUCTION Green leafy vegetables are a good source of important nutrients and nutraceuticals. In addition to containing 𝛽-carotene, a precursor of vitamin A, leafy vegetables are a cheap and sustainable source of vitamin C, chlorophyll and minerals.1,2 The fight against malnutrition and undernourishment continues to be a basic goal of development, and a variety of strategies, including those based on nutrient-rich foods such as leafy vegetables, is considered essential.3 Since most leafy vegetables are difficult to grow throughout the year and also their storage is challenging, different methods of drying have been an attractive alternative for usage of these perishable foods.4,5 Fresh leafy vegetables respire and transpire after harvesting and wilt, leading to loss of quality; in particular, there will be a loss of vitamins, which are very important for survival and play a crucial role in building the human body.6,7 The antioxidant activity of vitamin C can protect from damaging effects of air pollution and radiations, which will aid in preventing cancers.8 Seasonality of leafy vegetables can be overcome by suitable processing methods such as dehydration or drying and blanching of leaves. In spite of preservation, there is some minor loss of nutrients and pigments such as 𝛽-carotene and J Sci Food Agric (2014)
a major loss of vitamins due to the drying temperature and time required for vegetable processing.5,9 Apart from this, the nature of the solvent and the method of extraction influence the antioxidant potential of the resulting plant extract and need to be evaluated for each leafy vegetable before considering the same for food formulations.4,5 Previous studies pertaining to drying methods such as spray drying and fixed bed drying for roselle are limited to calyxes, wherein the retention of various bioactive compounds such as anthocyanins and phenolics and antioxidant activity has been addressed by various researchers.10 – 12 However, such studies are not available with respect to roselle foliage. Hibiscus sabdariffa L. (Malvaceae), roselle, is a common green leafy vegetable in southern states of India and some other parts of Asia.13 There are two types, based on their growth habit or
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Correspondence To: Parvatam Giridhar, Plant Cell Biotechnology Department, CSIR – Central Food Technological Research Institute, Mysore 570 020, India. E-mail: [email protected] Plant Cell Biotechnology Department, CSIR – Central Food Technological Research Institute, Mysore 570 020, India
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www.soci.org end use, namely H. sabdariffa var. sabdariffa and H. sabdariffa var. altissima Wester.14,15 Hibiscus sabdariffa is an annual or perennial shrub or woody sub-shrub growing to a height of 215–275 cm. The leaves have three to five lobes, a lamina diameter of 8–15 cm and a red petiole and stem.13,16 They are familiar as patwa (Hindi), yerra gongura (Telugu), pulichchai kerai (Tamil) and pundibija (Kannada).15 The leaves are mainly used for pickles, cooking and soup preparation because of their high iron and folic acid content.13 In addition, various health benefits such as diuretic, antiscorbutic, emollient and sedative effects are attributed to roselle.17 It is well known for its bast fibre content obtained from the plant stem and is used for various household purposes such as making sackcloth, twine and cord.18 The plant is rich in iron, potassium, ascorbic acid, fibre and 𝛽-carotene.19 In the present study the retention of chlorophyll pigment, ascorbic acid, total phenolic content, total flavonoid content and antioxidant compounds in H. sabdariffa leaves under seven different drying treatments (room-, sun-, oven-, microwave-, crossflow-, infrared- and freeze-drying methods) and the extraction efficiency of different solvents (methanol and 80% (v/v) alcohol), hot water and cold water were tested.
MATERIALS AND METHODS Plant material and chemicals Leaves of H. sabdariffa L. (roselle) were collected from local markets of Andhra Pradesh. A specimen of H. sabdariffa plant was deposited at the Herbarium Centre of the University of Mysore. The leaves were first graded and cleaned under running tap water, followed by their blotting on handmade filter paper to remove water. Subsequently, leaves were subjected to different drying methods. High-performance liquid chromatography (HPLC)-grade methanol, ethanol, acetone, ascorbic acid, gallic acid, 2,2diphenyl-1-picrylhydrazyl (DPPH) radical, metaphosphoric acid (MPA), glacial acetic acid, ethylenediaminetetraacetic acid (EDTA), sodium carbonate (Na2 CO3 ), Folin–Ciocalteu reagent, aluminium chloride (AlCl3 ), ammonium molybdate, potassium ferricyanide (K3 Fe(CN)6 ), 2.5 mol L−1 hydrochloric acid (HCl), phosphate buffer, sodium phosphate buffer, trichloroacetic acid (TCA) and ferric chloride (FeCl3 ) were obtained from Sisco Research Laboratory (Mumbai, India). All other chemicals used were of analytical grade. For HPLC analysis, degassed and 0.22 mm membrane-filtered triply distilled water was used. Drying methods Fresh leaves of H. sabdariffa (500 g each) were subjected to seven different drying methods, namely room (shade), sun, oven, microwave, crossflow, infrared and freeze drying. In shade drying, the leaves were spread over the room at 27 ± 2 ∘ C for 4 days for complete drying. In sun drying, the leaves were spread under sunlight at 35 ± 3 ∘ C midday temperature for 1 day. Oven drying (Ecocell, MMM Group, GmbH, Germany) involved drying the leaves at 65 ∘ C for 5 h. Microwave drying (M183DN with Triple Distribution System, Samsung, Bangkok, Thailand) was done at 850 W for 5 min. Crossflow drying (Technico Lab Products Pvt. Ltd., Chennai, India) was performed at 50 ± 5 ∘ C for 16 h. Infrared drying was done in a machine developed at CSIR-CFTRI, Mysore, India using 1.1–1.3 μm wavelength radiation at 50 ± 5 ∘ C for 5 h. In freeze drying, the leaves were lyophilized overnight on a vacuum tray at 0.012 mbar and −110 ∘ C in a freeze-dryer (CoolSafe™, Scanvac, Denmark). The dried leaf material was collected and ground
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(Maharaja Whiteline Perfect W&R 500 Mixer Grinder, Largo, Florida) at high speed for 20 min. The particle size of powdered leaf material was analysed (Blue Wave, Microtrac Inc., Largo, Florida, USA) to maintain a uniform particle size of about 250–270 μm for all dried leaves. Samples were stored in airtight polythene bags until further use. Determination of moisture content Briefly, 1 g of dried leaf material was heated in an oven at 50 ∘ C overnight and then cooled in a desiccator. The reduction in weight was calculated to obtain the final moisture content.20 Colour measurement The colour of dried leaf samples was monitored by measuring L, a and b values using a spectrophotometer (CM-5, Konica Minolta, Arlington, VA, USA). The L value represents lightness/darkness, the a value refers to red/green colour and the b value refers to blue/yellow colour.6 From the a and b values, values of chroma ((a2 + b2 )1/2 ) and hue angle (tan−1 (b/a)) were calculated. Determination of chlorophyll Dried leaf samples were extracted with 80% (v/v) acetone (1:10 w/v) using a mortar and pestle and centrifuged at 7000 × g for 10 min. The clear supernatant was collected and its absorbance (A) was measured at 661.5, 663, 645 and 450 nm with a double-beam spectrophotometer (UV-160 A, Shimadzu Corporation, Kyoto, Japan). Chlorophyll a (Chl.a), chlorophyll b (Chl.b) and total chlorophyll (Chl.t) concentrations were calculated according to Arnon21 as ( ) Chl.a μg mL−1 = 12.72A663 − 2.59A645 ( ) Chl.b μg mL−1 = 22.9A645 − 4.67A663 ( ) Chl.t μg mL−1 = 20.31A645 + 8.05A663 and according to Lichtenthaler22 as ( ) Chl.a μg mL−1 = 11.24A661.5 − 2.04A645 ( ) Chl.b μg mL−1 = 20.13A645 − 4.19A661.5 ( ) Chl.t μg mL−1 = 7.05A661.5 + 18.09A645 Extraction and estimation of ascorbic acid using HPLC Ascorbic acid was extracted (under subdued light) according to the method of Vanderslice et al.23 with some modification. A 1 g sample was homogenized with 2 mL of methanol and 10 mL of cold extraction solution containing 3% (w/v) MPA, 0.05% (w/v) EDTA and 0.8% (v/v) glacial acetic acid. After homogenization, the mixture was centrifuged at 3000 × g for 15 min at 4 ∘ C. The supernatant was collected and chromatographic analysis (LC-20AD, Shimadzu Corporation) of ascorbic acid was carried out. A Chromatopak C18 column (150 mm × 4.6 mm i.d., 5 μm particle size) was used for separation, with a mobile phase of 50 mmol L−1 K2 HPO4 adjusted to pH 7 (solvent A) and absolute methanol (solvent B). Elution was performed with 1% B for 5 min, a linear gradient from 1 to 30% B for 15 min, followed by 30% B for 10 min, at a flow rate of 1 mL min−1 . Ascorbic acid was monitored at 254 nm.
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Effect of drying methods on antioxidant potential of roselle foliage Sample extraction for in vitro antioxidant assays The extraction efficiency of different solvents, namely methanol and 80% (v/v) alcohol, as well as hot water and cold water was tested on dried leaves of H. sabdariffa. Briefly, 2.5 g of leaf powder was extracted with 50 mL of solvent using a mortar and pestle, shaken at 100 × g in a gyrorotary shaker for 30 min and centrifuged at 10 000 × g for 10 min. The pellet was collected and re-extracted by the same procedure. The two supernatants were pooled and stored in an amber tube to avoid light interference. All extractions and re-extractions were done in triplicate. Antioxidant assays Determination of total phenolic content The total phenolic content (TPC) of crude extracts was determined by the Folin–Ciocalteu method.24 A 0.2 mL aliquot of extract was pipetted into a test tube and the volume was made up to 3 mL with distilled water. Then 0.5 mL of Folin–Ciocalteu reagent was added and the mixture was incubated for 3 min, after which 2 mL of 20% (w/v) Na2 CO3 solution was added. The tube was vortexed and placed in boiling water bath for exactly 1 min. The tube contents were cooled and the absorbance at 650 nm was measured. The amount of phenolics present in each sample was determined using a previously plotted gallic acid standard graph. TPC was expressed as g gallic acid equivalent (GAE) kg−1 dry weight (DW) of leaf material. Determination of total flavonoid content The total flavonoid content (TFC) of each crude extract (0.1 mL) was diluted with absolute ethanol to an appropriate concentration. Then 1 mL of diluted sample was mixed with 1 mL of 2% (w/v) AlCl3 methanolic solution.25 After incubation at room temperature for 15 min, the absorbance of the reaction mixture at 430 nm was read with a double-beam spectrophotometer (UV-1800 A, Shimadzu Corporation). TFC was expressed as g rutin equivalent (RE) kg−1 DW of leaf material. Determination of total antioxidant activity by phosphomolybdenum method A 0.3 mL extract (2 mg mL−1 ) was combined with 28 mmol L−1 sodium phosphate and 4 mmol L−1 ammonium molybdate and incubated at 95 ∘ C for 90 min. After being cooled to room temperature, the absorbance of the solution at 695 nm was measured using a double-beam spectrophotometer (UV-160 A, Shimadzu Corporation).26 Total antioxidant activity (TAA) was expressed as g ascorbic acid equivalent (AAE) kg−1 DW of leaf material. DPPH free radical-scavenging activity assay Different dilutions of each extract (0.125–1.25 mg mL−1 ) were prepared in separate test tubes. Then 39.4 mg L−1 DPPH methanolic solution was added to each test tube to make up the total volume to 2 mL. The contents of the test tubes were mixed thoroughly and allowed to react for 15 min in the dark. The absorbance at 517 nm was measured with methanol as blank.27 From the absorbance, the inhibition or scavenging activity was calculated as DPPH − scavenging activity (%) ) ] [( = ODcontrol − ODsample ∕ODcontrol × 100 where OD denotes optical density. J Sci Food Agric (2014)
www.soci.org Ferric-reducing antioxidant power assay The ferric-reducing antioxidant power (FRAP) assay was done according to the method of Oyaizu.28 Different dilutions of each extract (1 mL) were mixed with 2.5 mL of 0.2 mol L−1 phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) K3 Fe (CN)6 and incubated at 50 ∘ C for 30 min. Then 2.5 mL of 10% (v/v) TCA was added and the mixture was centrifuged at 1000 × g for 10 min. Finally, 2.5 mL of the upper-layer solution was taken and mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% (w/v) FeCl3 . The procedure was carried out in triplicate and the absorbance at 700 nm was measured. The absorbance obtained was converted to g AAE kg−1 DW of leaf material. Statistical analysis Three parallel experiments were carried out for all analyses. All results are presented as mean ± standard deviation (SD) of three replicates. One-way analysis of variance (ANOVA) was performed using Microsoft Excel of Windows 7. Values with P < 0.05 were considered significant.
RESULTS AND DISCUSSION Moisture content The final moisture content of the seven differently dehydrated leaf materials ranged between about 2 and 9% (Fig. 1). The highest moisture content was observed in oven-dried samples (8.22%), while that of sun-dried samples was lowest (2.56%). These results are consistent with the findings for drumstick leaves.29 Colour It is important to observe the variation in colour (chroma value) of dried leaf materials, since it indicates the degradation of pigments such as chlorophyll. In both room- and freeze-dried samples the green colour of roselle leaves was significantly retained, as evidenced by the L, chroma and hue values (Fig. 2). Microwave drying had a drastic effect on colour, indicating extensive chlorophyll degradation. The chemical changes in pigments may be due to effects of temperature and oxidation during drying.6 Chlorophyll content The chlorophyll content of dried leaf materials is shown in Table 1. Thermal dehydration had greater degradation effects than non-thermal drying. Room and freeze drying led to maximum retention of chlorophyll. However, there was 40.2% degradation of chlorophyll in microwave drying, with Chl.a degraded more than Chl.b, followed by 34.91% in oven drying and 29.75% in crossflow drying. The necessity of comparison of chlorophyll estimation by the Arnon and Lichtenthaler methods was advocated in earlier reports.30,31 In the present study, where both methods were employed, there was a 10–16% increase in chlorophyll content quantified by the Arnon method compared with the Lichtenthaler method. Ascorbic acid content Ascorbic acid (vitamin C) is a very important vitamin that is involved in regulating blood pressure, contributes to reducing cholesterol levels and aids in the removal of cholesterol from arterial walls, thus preventing arteriosclerosis.32 The antioxidant property of vitamin C can also protect from damaging effects of air pollution and radiations and aid in preventing
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Figure 1. Influence of different drying treatments on moisture content of Hibiscus sabdariffa leaves. Values are mean ± SD of three replicates and significant at P < 0.05.
Figure 2. Colour parameters of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05. Table 1. Influence of different drying treatments on chlorophyll content (g kg−1 DW) of Hibiscus sabdariffa leaves Lichtenthaler method Sample RD SD OD MWD CFD IRD FD
Chl.a 1.12 ± 0.10 1.10 ± 0.02 0.83 ± 0.06 0.74 ± 0.04 0.86 ± 0.05 1.22 ± 0.12 1.15 ± 0.002
Chl.b 0.25 ± 0.01 0.20 ± 0.02 0.09 ± 0.01 0.11 ± 0.02 0.12 ± 0.02 0.19 ± 0.03 0.19 ± 0.02
Arnon method Chl.t 1.41 ± 0.003 1.30 ± 0.002 0.92 ± 0.01 0.85 ± 0.06 1.00 ± 0.05 1.34 ± 0.02 1.37 ± 0.11
Chl.a 1.32 ± 0.12 1.30 ± 0.03 0.99 ± 0.06 0.88 ± 0.05 1.01 ± 0.08 1.35 ± 0.01 1.34 ± 0.03
Chl.b 0.26 ± 0.005 0.20 ± 0.02 0.09 ± 0.01 0.13 ± 0.02 0.12 ± 0.02 0.19 ± 0.03 0.20 ± 0.03
Chl.t 1.59 ± 0.001 1.51 ± 0.05 1.08 ± 0.08 1.01 ± 0.07 1.14 ± 0.08 1.54 ± 0.08 1.55 ± 0.001
Values are mean ± SD of three replicates and significant at P < 0.05. RD, room-dried; SD, sun-dried; OD, oven-dried; MWD, microwave-dried; CFD, crossflow-dried; IRD, infrared-dried; FD, freeze-dried.
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Effect of drying methods on antioxidant potential of roselle foliage
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Figure 3. Ascorbic acid content (AAC) of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05.
cancers.8 The main function of ascorbic acid is in collagen synthesis and consequently in the formation and maintenance of cartilage, bones, gums, skin, teeth, etc.33 Van Duyne et al.34 reported 15–20% degradation of ascorbic acid during blanching and storage. Low-temperature drying of leafy vegetables was found to retain more 𝛽-carotene, vitamin C and chlorophyll than sun, solar or cabinet drying.1 Ascorbic acid retention depends mainly on the type of postharvest processing. During drying or dehydration, there will be more likelihood of ascorbic acid
degradation due to thermal and non-thermal treatments such as oxidative degradation.35 Vitamin C is the main compound contributing to antioxidant activities of green leaves and fruits.36 In the present study, room-dried H. sabdariffa leaf powder extracts had the highest content (11.11 g kg−1 DW) of ascorbic acid (Fig. 3). The order of retention of ascorbic acid in different samples was room-dried > freeze-dried > infrared-dried > crossflow-dried > oven-dried > sun-dried > microwave-dried. In view of the various health benefits attributed to it, the retention of significant
25 Methanol
80% Alcohol
Hot water
Cold water
g kg–1 GAE
20
15
10
5
0
Room
Sun
Oven
Microoven Cross flow
Infra red Freeze dried
Drying method Figure 4. Total phenolic content (TPC) of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05.
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25 Methanol
80% Alcohol
Hot water
Cold water
g kg–1 RE
20
15
10
5
0
Room
Sun
Oven
Microoven Cross flow
Infra red Freeze dried
Drying method Figure 5. Total flavonoid content (TFC) of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05.
levels of ascorbic acid during food processing is of paramount importance.33 Antioxidant properties Total phenolic content Phenolics are aromatic secondary plant metabolites widely spread throughout the plant kingdom and associated with colour, sensory qualities and nutritional and antioxidant properties of foods. The antioxidant property of phenolics is due to their
redox properties.37 The TPC obtained with different methods of drying and different solvents is shown in Fig. 4. It can be observed that the TPC varied with different solvent extractions and different drying methods. The room-dried leaf cold water extract had a higher TPC content (19.39 g GAE kg−1 DW) than the corresponding organic solvent and hot water extracts, which was further supported by similar observations in Phyllanthus amarus.38 Methanol extracts of fresh samples have a higher TPC than those of processed samples, because methanol can denature
Figure 6. Total antioxidant activity (TAA) of differently dried leaves of Hibiscus sabdariffa. Values are mean ± SD of three replicates and significant at P < 0.05.
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Effect of drying methods on antioxidant potential of roselle foliage
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polyphenol oxidases in plant cell wall degradation and thus extract more TPC than water. Not only in leafy vegetables but also in fresh fruits has the superiority of methanol over other solvents for TPC extraction been reported.2,36,39 However, processed plant material extracted with methanol will have lower TPC and antioxidant properties than that extracted with water.38 In dried plant materials, polar compounds are extracted more into polar solvents such as water. It has been proposed that hot water extraction is better than cold water extraction, as there will be a release of cell wall phenolics in the case of hot water extraction.40 However, the order of extractability of TPC in our study was cold water > hot water > 80% alcohol > methanol.
Total antioxidant activity Different sample extracts were used to determine their antioxidant capacities by the formation of phosphomolybdenum complexes. The method is based on the reduction of Mo(VI) to Mo(V) by antioxidant compounds and the formation of a green Mo(V) complex with maximum absorbance at 695 nm. Leaf material of H. sabdariffa dried by different methods exhibited various degrees of antioxidant capacity (Fig. 6). The sun-dried leaf cold water extract showed the highest TAA (69.46 g AAE kg−1 DW), followed by the room-dried leaf 80% alcohol extract (65.01 g AAE kg−1 DW). Variations in antioxidant activity may be due to different phenolic, flavonoid and ascorbic acid contents.
Total flavonoid content Flavonoid compounds are more extractable in organic solvents than in water. The room-dried leaf methanol extract exhibited the highest flavonoid content (19.09 g RE kg−1 DW), followed by the freeze-dried leaf 80% alcohol extract (18.66 g RE kg−1 DW g kg−1 ) (Fig. 5). In addition, the crossflow-dried leaf 80% alcohol extract had a higher flavonoid content (12.12 g RE kg−1 DW) than the room-dried leaf hot and cold water extracts (8.57 and 7.06 g RE kg−1 DW respectively) and the oven-dried leaf hot and cold water extracts (3.21 and 3.13 g RE kg−1 DW respectively). The order of extractability of flavonoids was methanol > 80% alcohol > hot water > cold water.
DPPH free radical-scavenging activity The DPPH free radical-scavenging activity assay is most widely used to determine the primary antioxidant activity of antioxidant compounds in plant and fruit extracts.27 The method is based on the reduction of DPPH radicals in methanol, which causes a drop in absorbance at 517 nm. In this study, ascorbic acid was used as standard antioxidant. The DPPH free radical-scavenging activity of different sample extracts is shown in Fig. 7. All freeze-dried leaf extracts showed higher scavenging activity (cold water 93.7% and hot water 91.7% at 1.25 mg mL−1 and methanol 89.47% and aqueous alcohol 83.47% at 0.625 mg mL−1 ) than ascorbic acid (84.12% at 15 μg mL−1 ). Similarly, the variation in antioxidant
Figure 7. DPPH free radical-scavenging activity of room-dried (RD), sun-dried (SD), oven-dried (OD), microwave-dried (MWD), crossflow-dried (CFD), infrared-dried (IRD) and freeze-dried (FD) leaves of Hibiscus sabdariffa (AA, ascorbic acid standard): a, methanol extracts; b, 80% (v/v) aqueous alcohol extracts; c, hot water extracts; d, cold water extracts. Values are mean ± SD of three replicates and significant at P < 0.05.
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Figure 8. Ferric-reducing antioxidant power (FRAP) of differently dried leaves of Hibiscus sabdariffa. Values are mean ± SD of three replicates and significant at P < 0.05.
properties of ginger leaves dried by different methods has been studied.4 Although phenols were reported to contribute most towards antioxidant activity, a strong correlation between phenol and pigment contents was also observed.27 Ferric-reducing antioxidant power The ability of the dried leaf extracts to reduce ferric ions was evident and varied with the extraction solvent and drying method used (Fig. 8). The freeze-dried leaf 80% alcohol extract exhibited higher FRAP (35.74 ± 0.87 g AAE kg−1 DW) than the corresponding methanol, cold water and hot water extracts. Iron in ferrous form is common in food systems and its high redox potential contributes to oxidative stress at the cellular level, leading to various physiological dysfunctions.41 The purpose of the test for ferrous ion-chelating activity was to determine the capacity of dried leaf extracts of H. sabdariffa to bind the ferrous ion catalysing oxidation. In this assay the leaf extracts showed chelating activity by capturing ferrous ions before ferrozine. A similar observation was reported for other leafy vegetables.42 Owing to the efficient FRAP of leaf extracts in this study, due to their TPC, TFC and TAA, the postharvest processed leaves of H. sabdariffa can be investigated further systematically to ascertain the level of contribution in protecting cells from metal-induced oxidative stress, with the aim of further supporting H. sabdariffa-fortified food formulations as functional foods. Discussion Overall, different drying methods showed a moderate to low effect on TPC and TFC, indicating that TPC and TFC are rather stable to different dehydration conditions. The low retention of nutrients and antioxidant activity in infrared-, oven- and microwave-dried leaves may have been caused by enzymatic processes, which can lead to substantial changes in the composition of phytochemicals
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and in antioxidant activity.43 Similarly, different drying methods exhibited a profound influence on ascorbic acid content, and all thermal treatments have been reported to significantly decrease the total flavonoid content in soybeans.44 Only 25% of ascorbic acid was retained in sun- and microwave-dried leaf samples, in agreement with similar studies on leaves of Moringa oleifera.5 A number of dehydration procedures such as sun, crossflow, drum, spray, puff, freeze and microwave drying are employed for fruits and vegetables.5 In general, for most vegetables, drying leads to 10–20% loss of pigments such as carotenoids, with the increased surface area of dried or powdered products leading to further losses (through autoxidation) unless they are protected from air and light. Some reports have explained the influence of drying methods on roselle calyxes, wherein the effect of temperature on spray drying of roselle calyx extracts and the retention of monomeric anthocyanins, phenolic compounds, polymeric colour and antioxidant activity were observed upon performing dehydration using fixed bed dryer equipment.11,12 In a recent study the bioactive compound retention potential of aqueous extracts of roselle calyxes and its usage in a dairy beverage product were demonstrated.10
CONCLUSION Room and freeze drying were the best methods for retention of chlorophyll, ascorbic acid and antioxidant compounds of H. sabdariffa leaves. Other methods such as microwave, oven, sun, crossflow and infrared drying had a significant impact on TPC, TFC and TAA. Similarly, water extracts exhibited higher TPC and TAA than organic solvent extracts. The results of this study have implications for H. sabdariffa leaf powder-based processed food formulations in view of its potential quality parameters being retained under suitable drying methods.
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Effect of drying methods on antioxidant potential of roselle foliage
ACKNOWLEDGEMENTS The authors are thankful to the Department of Biotechnology, Government of India, New Delhi for financial assistance. We greatly acknowledge the Director, CSIR-CFTRI for his kind support.
REFERENCES 1 Negi PS and Roy SK, Effect of blanching and drying methods on 𝛽-carotene, ascorbic acid and chlorophyll retention of leafy vegetables. LWT – Food Sci Technol 33:295–298 (2000). 2 Saini RK, Shetty NP and Giridhar P, Carotenoid content in vegetative and reproductive parts of commercially grown Moringa oleifera Lam. cultivars from India by LC–APCI–MS. Eur Food Res Technol 238:971–978 (2014). 3 Yadav SK and Sehgal S, Effect of home processing on ascorbic acid and 𝛽-carotene content of spinach (Spinacia oleracea) and amaranth (Amaranthus tricolor) leaves. Plant Foods Hum Nutr 47:125–131 (1995). 4 Chan EWC, Lim YY, Wong SK, Lim KK, Tan SP, Lianto FS, et al., Effect of different drying methods on the antioxidant properties of leaves and tea of ginger species. Food Chem 113:166–172 (2009). 5 Saini RK, Shetty NP, Prakash M and Giridhar P, Effect of dehydration methods on retention of carotenoids, tocopherols, ascorbic acid and antioxidant activity in Moringa oleifera leaves and preparation of a RTE product. J Food Sci Technol 51:2176–2182 (2014). 6 Shitanda D and Wanjala NV, Effect of different drying methods on the quality of jute (Corchorus olitorius L.). Dry Technol 24:95–98 (2006). 7 Sarkiyayi S and Ikioda H, Estimation of thiamin and ascorbic acid contents in fresh and dried Hibiscus sabdariffa (roselle) and Lactuca sativa (lettuce). Adv J Food Sci Technol 2:47–49 (2010). 8 Frederick R and Klenner MD, Observation on the dose of vitamins and administration of ascorbic acid. J Appl Nutr 23:2–3 (1971). 9 Speek AJ, Speek SS and Schreurs WHP, Total carotenoid and carotene contents of Thai vegetables and the effect of processing. Food Chem 27:245–257 (1988). 10 Leyva Daniel DE, Barragán Huerta BE, Vizcarra Mendoza MG and Anaya Sosa I, Effect of drying conditions on the retention of phenolic compounds, anthocyanins and antioxidant activity of roselle (Hibiscus sabdariffa L.) added to yogurt. Int J Food Sci Technol 48:2283–2291 (2013). 11 Leyva Daniel D, Barragán Huerta BE, Anaya Sosa I and Vizcarra Mendoza MG, Effect of fixed bed drying on the retention of phenolic compounds, anthocyanins and antioxidant activity of roselle (Hibiscus sabdariffa L.). Ind Crops Prod 40:268–276 (2012). 12 Gonzalez-Palomares S, Estarron-Espinosa M, Gomez-Leyva JF and Andrade-Gonzalez I, Effect of the temperature on the spray drying of roselle extracts (Hibiscus sabdariffa L.). Plant Foods Hum Nutr 64:62–67 (2009). 13 Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products, Vol. V, Raw Materials. Council of Scientific and Industrial Research, New Delhi (1959). 14 Ismail A, Ikram EHK and Nazir HSM, Roselle (Hibiscus sabdariffa L.) seeds – nutritional composition, protein quality and health benefits. Food 2:1–16 (2008). 15 Mahadevan NS and Pradeep K, Hibiscus sabdariffa Linn. – an overview. Nat Prod Rad 8:77–83 (2009). 16 Asaolu SS, Adefemi OS, Oyakilome IG, Ajibulu KE and Asaolu MF, Proximate and mineral composition of Nigerian leafy vegetables. J Food Res 1:214–218 (2012). 17 Komarov VL (ed.), Flora of the USSR (English Translation). Israel Program for Scientific Translation, Jerusalem (1968). 18 Arvind M and Alka C, Hibiscus sabdariffa L. A rich source of secondary metabolites. Int J Pharmaceut Sci Rev Res 6:83–87 (2011). 19 Duke JA and Atchley AA, Proximate analysis, in The Handbook of Plant Science in Agriculture, ed. by Christie BR. CRC Press, Boca Raton, FL (1984). 20 AOCS, Official Methods and Recommended Practices of the American Oil Chemists’ Society (5th edn). AOCS Press, Champaign, IL (2003). 21 Arnon DI, Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. J Plant Physiol 24:1–15 (1949).
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www.soci.org 22 Lichtenthaler HK, Vegetation stress: an introduction to the stress concept in plants. J Plant Physiol 148:4–14 (1996). 23 Vanderslice JT, Higgs DJ, Hayes JM and Block G, Ascorbic acid and dehydroascorbic acid content of foods-as-eaten. J Food Compos Anal 3:105–118 (1990). 24 Sadasivam S and Manickam A, Biochemical Methods (8th edn). New Age International, New Delhi (2008). 25 Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P and Vidal N, Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds. Food Chem 97:654–660 (2006). 26 Prieto P, Pineda M and Agulilar M, Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem 269:337–341 (1999). 27 Khan MI, Sri Harsha PSC, Giridhar P and Ravishankar GA, Pigment identification, nutritional composition, bioactivity, and in vitro cell cytotoxicity of Rivina humilis L. berries, potential source of betalains. LWT – Food Sci Technol 47:315–323 (2012). 28 Oyaizu M, Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine. Jpn J Nutr 44:307–315 (1986). 29 Satwase AN, Pandhre GR, Sirsat PG and Wade YR, Studies on drying characteristic and nutritional composition of drumstick leaves by using sun, shadow, cabinet and oven drying methods. Sci Rep 2:584–588 (2013). 30 Wellburn AR, The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313 (1994). 31 Porra RJ, The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73:149–156 (2002). 32 Addo AA, Seasonal availability of dietary ascorbic acid and incidence of scurvy in northern state of Nigeria. PhD Thesis, Department of Biochemistry, Ahmadu Bello University, Zaria (2004). 33 Davey MW, Montagu MV, Inze D, Sanmartin M, Kanellis A, Smirnoff N, et al. Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Sci Food Agric 80:825–860 (2000). 34 Van Duyne F, Buckart S, Chase J and Simpson J, Ascorbic acid content of freshly harvested vegetables. J Am Diet Assoc 21:153–154 (1945). 35 Sagar VR and Suresh Kumar P, Recent advances in drying and dehydration of fruits and vegetables: a review. J Food Sci Technol 47:15–26 (2010). 36 Khan MI, Sri Harsha PSC, Giridhar P and Ravishankar GA, Pigment identification, antioxidant activity, and nutrient composition of Tinospora cordifolia (Willd.) Miers ex Hook. f & Thoms fruit. Int J Food Sci Nutr 62:239–249 (2011). 37 Kaur C and Kapoor HC, Antioxidant activity and total phenolic content of some Asian vegetables. Int J Food Sci Technol 37:153–161 (2002). 38 Lim YY and Murtijaya J, Antioxidant properties of Phyllanthus amarus extracts as affected by different drying methods. LWT – Food Sci Technol 40:1664–1669 (2007). 39 Sri Harsha PSC, Khan MI, Prabhakar P and Giridhar P, Cyanidin-3-glucoside, nutritionally important constituents and in vitro antioxidant activities of Santalum album L. berries. Food Res Int 50:275–281 (2013). 40 Toor RK and Savage GP, Effect of semi-drying on the antioxidant compounds of tomatoes. Food Chem 94:90–97 (2006). 41 Gandia-Herrero F, Escribano J and Garcia-Carmona F, The role of phenolic hydroxyl groups in the free radical scavenging activity of betalains. J Nat Prod 72:1142–1146 (2009). 42 Gupta S and Prakash J, Studies on Indian green leafy vegetables for their antioxidants activity. Plant Foods Hum Nutr 64:39–45 (2009). 43 Tomaino A, Cimino F, Zimbalatti V, Venuti V, Sulfaro V and De Pasquale A, Influence of heating on antioxidant activity and the chemical composition of some spice essential oils. Food Chem 89:549–554 (2005). 44 Yang HW, Hsu CK and Yang YF, Effect of thermal treatments on anti-nutritional factors and antioxidant capabilities in yellow soybeans and green-cotyledon small black soybeans. J Sci Food Agric 94:1794–1801 (2014).
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Revised: 13 August 2014
Accepted article published: 20 August 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6879
Effect of different drying methods on chlorophyll, ascorbic acid and antioxidant compounds retention of leaves of Hibiscus sabdariffa L. Sandopu Sravan Kumar, Prabhakaran Manoj, Nandini P Shetty and Parvatam Giridhar* Abstract BACKGROUND: Use of the indigenous, easily accessible leafy vegetable roselle (Hibiscus sabdariffa L.) for value addition is gaining impetus as its nutritive and nutraceutical compounds are exposed by investigations. Being a perishable, storage is challenging, hence different methods of drying have been an attractive alternative for its postharvest usage in foods without much compromising its quality and antioxidant potential. RESULTS: Room- and freeze-dried samples were found to have best quality in terms of colour, total flavonoid content (18.53 ± 2.39 and 18.66 ± 1.06 g kg−1 respectively), total phenolic content (17.76 ± 1.93 and 18.91 ± 0.48 g kg−1 ), chlorophyll content (1.59 ± 0.001 and 1.55 ± 0.001 g kg−1 ) and ascorbic acid content (11.11 ± 1.04 and 8.92 ± 0.94 g kg−1 ) compared with those subjected to infrared, crossflow, microwave, oven or sun drying. Samples treated by room and freeze drying retained maximum antioxidant potential as shown by the phosphomolybdate method and the 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity and ferric-reducing antioxidant power assays. Cold water and hot water extracts showed significantly higher total phenolic content and total antioxidant activity owing to the greater solubility of phenolics and destruction of cellular components in polar solvents than in organic solvents. CONCLUSION: The data obtained show the potential for retaining quality parameters of roselle leaf under suitable drying methods. © 2014 Society of Chemical Industry Keywords: roselle; drying methods; ascorbic acid; antioxidant properties; sun drying; microwave drying
INTRODUCTION Green leafy vegetables are a good source of important nutrients and nutraceuticals. In addition to containing 𝛽-carotene, a precursor of vitamin A, leafy vegetables are a cheap and sustainable source of vitamin C, chlorophyll and minerals.1,2 The fight against malnutrition and undernourishment continues to be a basic goal of development, and a variety of strategies, including those based on nutrient-rich foods such as leafy vegetables, is considered essential.3 Since most leafy vegetables are difficult to grow throughout the year and also their storage is challenging, different methods of drying have been an attractive alternative for usage of these perishable foods.4,5 Fresh leafy vegetables respire and transpire after harvesting and wilt, leading to loss of quality; in particular, there will be a loss of vitamins, which are very important for survival and play a crucial role in building the human body.6,7 The antioxidant activity of vitamin C can protect from damaging effects of air pollution and radiations, which will aid in preventing cancers.8 Seasonality of leafy vegetables can be overcome by suitable processing methods such as dehydration or drying and blanching of leaves. In spite of preservation, there is some minor loss of nutrients and pigments such as 𝛽-carotene and J Sci Food Agric (2014)
a major loss of vitamins due to the drying temperature and time required for vegetable processing.5,9 Apart from this, the nature of the solvent and the method of extraction influence the antioxidant potential of the resulting plant extract and need to be evaluated for each leafy vegetable before considering the same for food formulations.4,5 Previous studies pertaining to drying methods such as spray drying and fixed bed drying for roselle are limited to calyxes, wherein the retention of various bioactive compounds such as anthocyanins and phenolics and antioxidant activity has been addressed by various researchers.10 – 12 However, such studies are not available with respect to roselle foliage. Hibiscus sabdariffa L. (Malvaceae), roselle, is a common green leafy vegetable in southern states of India and some other parts of Asia.13 There are two types, based on their growth habit or
∗
Correspondence To: Parvatam Giridhar, Plant Cell Biotechnology Department, CSIR – Central Food Technological Research Institute, Mysore 570 020, India. E-mail: [email protected] Plant Cell Biotechnology Department, CSIR – Central Food Technological Research Institute, Mysore 570 020, India
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www.soci.org end use, namely H. sabdariffa var. sabdariffa and H. sabdariffa var. altissima Wester.14,15 Hibiscus sabdariffa is an annual or perennial shrub or woody sub-shrub growing to a height of 215–275 cm. The leaves have three to five lobes, a lamina diameter of 8–15 cm and a red petiole and stem.13,16 They are familiar as patwa (Hindi), yerra gongura (Telugu), pulichchai kerai (Tamil) and pundibija (Kannada).15 The leaves are mainly used for pickles, cooking and soup preparation because of their high iron and folic acid content.13 In addition, various health benefits such as diuretic, antiscorbutic, emollient and sedative effects are attributed to roselle.17 It is well known for its bast fibre content obtained from the plant stem and is used for various household purposes such as making sackcloth, twine and cord.18 The plant is rich in iron, potassium, ascorbic acid, fibre and 𝛽-carotene.19 In the present study the retention of chlorophyll pigment, ascorbic acid, total phenolic content, total flavonoid content and antioxidant compounds in H. sabdariffa leaves under seven different drying treatments (room-, sun-, oven-, microwave-, crossflow-, infrared- and freeze-drying methods) and the extraction efficiency of different solvents (methanol and 80% (v/v) alcohol), hot water and cold water were tested.
MATERIALS AND METHODS Plant material and chemicals Leaves of H. sabdariffa L. (roselle) were collected from local markets of Andhra Pradesh. A specimen of H. sabdariffa plant was deposited at the Herbarium Centre of the University of Mysore. The leaves were first graded and cleaned under running tap water, followed by their blotting on handmade filter paper to remove water. Subsequently, leaves were subjected to different drying methods. High-performance liquid chromatography (HPLC)-grade methanol, ethanol, acetone, ascorbic acid, gallic acid, 2,2diphenyl-1-picrylhydrazyl (DPPH) radical, metaphosphoric acid (MPA), glacial acetic acid, ethylenediaminetetraacetic acid (EDTA), sodium carbonate (Na2 CO3 ), Folin–Ciocalteu reagent, aluminium chloride (AlCl3 ), ammonium molybdate, potassium ferricyanide (K3 Fe(CN)6 ), 2.5 mol L−1 hydrochloric acid (HCl), phosphate buffer, sodium phosphate buffer, trichloroacetic acid (TCA) and ferric chloride (FeCl3 ) were obtained from Sisco Research Laboratory (Mumbai, India). All other chemicals used were of analytical grade. For HPLC analysis, degassed and 0.22 mm membrane-filtered triply distilled water was used. Drying methods Fresh leaves of H. sabdariffa (500 g each) were subjected to seven different drying methods, namely room (shade), sun, oven, microwave, crossflow, infrared and freeze drying. In shade drying, the leaves were spread over the room at 27 ± 2 ∘ C for 4 days for complete drying. In sun drying, the leaves were spread under sunlight at 35 ± 3 ∘ C midday temperature for 1 day. Oven drying (Ecocell, MMM Group, GmbH, Germany) involved drying the leaves at 65 ∘ C for 5 h. Microwave drying (M183DN with Triple Distribution System, Samsung, Bangkok, Thailand) was done at 850 W for 5 min. Crossflow drying (Technico Lab Products Pvt. Ltd., Chennai, India) was performed at 50 ± 5 ∘ C for 16 h. Infrared drying was done in a machine developed at CSIR-CFTRI, Mysore, India using 1.1–1.3 μm wavelength radiation at 50 ± 5 ∘ C for 5 h. In freeze drying, the leaves were lyophilized overnight on a vacuum tray at 0.012 mbar and −110 ∘ C in a freeze-dryer (CoolSafe™, Scanvac, Denmark). The dried leaf material was collected and ground
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(Maharaja Whiteline Perfect W&R 500 Mixer Grinder, Largo, Florida) at high speed for 20 min. The particle size of powdered leaf material was analysed (Blue Wave, Microtrac Inc., Largo, Florida, USA) to maintain a uniform particle size of about 250–270 μm for all dried leaves. Samples were stored in airtight polythene bags until further use. Determination of moisture content Briefly, 1 g of dried leaf material was heated in an oven at 50 ∘ C overnight and then cooled in a desiccator. The reduction in weight was calculated to obtain the final moisture content.20 Colour measurement The colour of dried leaf samples was monitored by measuring L, a and b values using a spectrophotometer (CM-5, Konica Minolta, Arlington, VA, USA). The L value represents lightness/darkness, the a value refers to red/green colour and the b value refers to blue/yellow colour.6 From the a and b values, values of chroma ((a2 + b2 )1/2 ) and hue angle (tan−1 (b/a)) were calculated. Determination of chlorophyll Dried leaf samples were extracted with 80% (v/v) acetone (1:10 w/v) using a mortar and pestle and centrifuged at 7000 × g for 10 min. The clear supernatant was collected and its absorbance (A) was measured at 661.5, 663, 645 and 450 nm with a double-beam spectrophotometer (UV-160 A, Shimadzu Corporation, Kyoto, Japan). Chlorophyll a (Chl.a), chlorophyll b (Chl.b) and total chlorophyll (Chl.t) concentrations were calculated according to Arnon21 as ( ) Chl.a μg mL−1 = 12.72A663 − 2.59A645 ( ) Chl.b μg mL−1 = 22.9A645 − 4.67A663 ( ) Chl.t μg mL−1 = 20.31A645 + 8.05A663 and according to Lichtenthaler22 as ( ) Chl.a μg mL−1 = 11.24A661.5 − 2.04A645 ( ) Chl.b μg mL−1 = 20.13A645 − 4.19A661.5 ( ) Chl.t μg mL−1 = 7.05A661.5 + 18.09A645 Extraction and estimation of ascorbic acid using HPLC Ascorbic acid was extracted (under subdued light) according to the method of Vanderslice et al.23 with some modification. A 1 g sample was homogenized with 2 mL of methanol and 10 mL of cold extraction solution containing 3% (w/v) MPA, 0.05% (w/v) EDTA and 0.8% (v/v) glacial acetic acid. After homogenization, the mixture was centrifuged at 3000 × g for 15 min at 4 ∘ C. The supernatant was collected and chromatographic analysis (LC-20AD, Shimadzu Corporation) of ascorbic acid was carried out. A Chromatopak C18 column (150 mm × 4.6 mm i.d., 5 μm particle size) was used for separation, with a mobile phase of 50 mmol L−1 K2 HPO4 adjusted to pH 7 (solvent A) and absolute methanol (solvent B). Elution was performed with 1% B for 5 min, a linear gradient from 1 to 30% B for 15 min, followed by 30% B for 10 min, at a flow rate of 1 mL min−1 . Ascorbic acid was monitored at 254 nm.
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Effect of drying methods on antioxidant potential of roselle foliage Sample extraction for in vitro antioxidant assays The extraction efficiency of different solvents, namely methanol and 80% (v/v) alcohol, as well as hot water and cold water was tested on dried leaves of H. sabdariffa. Briefly, 2.5 g of leaf powder was extracted with 50 mL of solvent using a mortar and pestle, shaken at 100 × g in a gyrorotary shaker for 30 min and centrifuged at 10 000 × g for 10 min. The pellet was collected and re-extracted by the same procedure. The two supernatants were pooled and stored in an amber tube to avoid light interference. All extractions and re-extractions were done in triplicate. Antioxidant assays Determination of total phenolic content The total phenolic content (TPC) of crude extracts was determined by the Folin–Ciocalteu method.24 A 0.2 mL aliquot of extract was pipetted into a test tube and the volume was made up to 3 mL with distilled water. Then 0.5 mL of Folin–Ciocalteu reagent was added and the mixture was incubated for 3 min, after which 2 mL of 20% (w/v) Na2 CO3 solution was added. The tube was vortexed and placed in boiling water bath for exactly 1 min. The tube contents were cooled and the absorbance at 650 nm was measured. The amount of phenolics present in each sample was determined using a previously plotted gallic acid standard graph. TPC was expressed as g gallic acid equivalent (GAE) kg−1 dry weight (DW) of leaf material. Determination of total flavonoid content The total flavonoid content (TFC) of each crude extract (0.1 mL) was diluted with absolute ethanol to an appropriate concentration. Then 1 mL of diluted sample was mixed with 1 mL of 2% (w/v) AlCl3 methanolic solution.25 After incubation at room temperature for 15 min, the absorbance of the reaction mixture at 430 nm was read with a double-beam spectrophotometer (UV-1800 A, Shimadzu Corporation). TFC was expressed as g rutin equivalent (RE) kg−1 DW of leaf material. Determination of total antioxidant activity by phosphomolybdenum method A 0.3 mL extract (2 mg mL−1 ) was combined with 28 mmol L−1 sodium phosphate and 4 mmol L−1 ammonium molybdate and incubated at 95 ∘ C for 90 min. After being cooled to room temperature, the absorbance of the solution at 695 nm was measured using a double-beam spectrophotometer (UV-160 A, Shimadzu Corporation).26 Total antioxidant activity (TAA) was expressed as g ascorbic acid equivalent (AAE) kg−1 DW of leaf material. DPPH free radical-scavenging activity assay Different dilutions of each extract (0.125–1.25 mg mL−1 ) were prepared in separate test tubes. Then 39.4 mg L−1 DPPH methanolic solution was added to each test tube to make up the total volume to 2 mL. The contents of the test tubes were mixed thoroughly and allowed to react for 15 min in the dark. The absorbance at 517 nm was measured with methanol as blank.27 From the absorbance, the inhibition or scavenging activity was calculated as DPPH − scavenging activity (%) ) ] [( = ODcontrol − ODsample ∕ODcontrol × 100 where OD denotes optical density. J Sci Food Agric (2014)
www.soci.org Ferric-reducing antioxidant power assay The ferric-reducing antioxidant power (FRAP) assay was done according to the method of Oyaizu.28 Different dilutions of each extract (1 mL) were mixed with 2.5 mL of 0.2 mol L−1 phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) K3 Fe (CN)6 and incubated at 50 ∘ C for 30 min. Then 2.5 mL of 10% (v/v) TCA was added and the mixture was centrifuged at 1000 × g for 10 min. Finally, 2.5 mL of the upper-layer solution was taken and mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% (w/v) FeCl3 . The procedure was carried out in triplicate and the absorbance at 700 nm was measured. The absorbance obtained was converted to g AAE kg−1 DW of leaf material. Statistical analysis Three parallel experiments were carried out for all analyses. All results are presented as mean ± standard deviation (SD) of three replicates. One-way analysis of variance (ANOVA) was performed using Microsoft Excel of Windows 7. Values with P < 0.05 were considered significant.
RESULTS AND DISCUSSION Moisture content The final moisture content of the seven differently dehydrated leaf materials ranged between about 2 and 9% (Fig. 1). The highest moisture content was observed in oven-dried samples (8.22%), while that of sun-dried samples was lowest (2.56%). These results are consistent with the findings for drumstick leaves.29 Colour It is important to observe the variation in colour (chroma value) of dried leaf materials, since it indicates the degradation of pigments such as chlorophyll. In both room- and freeze-dried samples the green colour of roselle leaves was significantly retained, as evidenced by the L, chroma and hue values (Fig. 2). Microwave drying had a drastic effect on colour, indicating extensive chlorophyll degradation. The chemical changes in pigments may be due to effects of temperature and oxidation during drying.6 Chlorophyll content The chlorophyll content of dried leaf materials is shown in Table 1. Thermal dehydration had greater degradation effects than non-thermal drying. Room and freeze drying led to maximum retention of chlorophyll. However, there was 40.2% degradation of chlorophyll in microwave drying, with Chl.a degraded more than Chl.b, followed by 34.91% in oven drying and 29.75% in crossflow drying. The necessity of comparison of chlorophyll estimation by the Arnon and Lichtenthaler methods was advocated in earlier reports.30,31 In the present study, where both methods were employed, there was a 10–16% increase in chlorophyll content quantified by the Arnon method compared with the Lichtenthaler method. Ascorbic acid content Ascorbic acid (vitamin C) is a very important vitamin that is involved in regulating blood pressure, contributes to reducing cholesterol levels and aids in the removal of cholesterol from arterial walls, thus preventing arteriosclerosis.32 The antioxidant property of vitamin C can also protect from damaging effects of air pollution and radiations and aid in preventing
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Figure 1. Influence of different drying treatments on moisture content of Hibiscus sabdariffa leaves. Values are mean ± SD of three replicates and significant at P < 0.05.
Figure 2. Colour parameters of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05. Table 1. Influence of different drying treatments on chlorophyll content (g kg−1 DW) of Hibiscus sabdariffa leaves Lichtenthaler method Sample RD SD OD MWD CFD IRD FD
Chl.a 1.12 ± 0.10 1.10 ± 0.02 0.83 ± 0.06 0.74 ± 0.04 0.86 ± 0.05 1.22 ± 0.12 1.15 ± 0.002
Chl.b 0.25 ± 0.01 0.20 ± 0.02 0.09 ± 0.01 0.11 ± 0.02 0.12 ± 0.02 0.19 ± 0.03 0.19 ± 0.02
Arnon method Chl.t 1.41 ± 0.003 1.30 ± 0.002 0.92 ± 0.01 0.85 ± 0.06 1.00 ± 0.05 1.34 ± 0.02 1.37 ± 0.11
Chl.a 1.32 ± 0.12 1.30 ± 0.03 0.99 ± 0.06 0.88 ± 0.05 1.01 ± 0.08 1.35 ± 0.01 1.34 ± 0.03
Chl.b 0.26 ± 0.005 0.20 ± 0.02 0.09 ± 0.01 0.13 ± 0.02 0.12 ± 0.02 0.19 ± 0.03 0.20 ± 0.03
Chl.t 1.59 ± 0.001 1.51 ± 0.05 1.08 ± 0.08 1.01 ± 0.07 1.14 ± 0.08 1.54 ± 0.08 1.55 ± 0.001
Values are mean ± SD of three replicates and significant at P < 0.05. RD, room-dried; SD, sun-dried; OD, oven-dried; MWD, microwave-dried; CFD, crossflow-dried; IRD, infrared-dried; FD, freeze-dried.
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Effect of drying methods on antioxidant potential of roselle foliage
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Figure 3. Ascorbic acid content (AAC) of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05.
cancers.8 The main function of ascorbic acid is in collagen synthesis and consequently in the formation and maintenance of cartilage, bones, gums, skin, teeth, etc.33 Van Duyne et al.34 reported 15–20% degradation of ascorbic acid during blanching and storage. Low-temperature drying of leafy vegetables was found to retain more 𝛽-carotene, vitamin C and chlorophyll than sun, solar or cabinet drying.1 Ascorbic acid retention depends mainly on the type of postharvest processing. During drying or dehydration, there will be more likelihood of ascorbic acid
degradation due to thermal and non-thermal treatments such as oxidative degradation.35 Vitamin C is the main compound contributing to antioxidant activities of green leaves and fruits.36 In the present study, room-dried H. sabdariffa leaf powder extracts had the highest content (11.11 g kg−1 DW) of ascorbic acid (Fig. 3). The order of retention of ascorbic acid in different samples was room-dried > freeze-dried > infrared-dried > crossflow-dried > oven-dried > sun-dried > microwave-dried. In view of the various health benefits attributed to it, the retention of significant
25 Methanol
80% Alcohol
Hot water
Cold water
g kg–1 GAE
20
15
10
5
0
Room
Sun
Oven
Microoven Cross flow
Infra red Freeze dried
Drying method Figure 4. Total phenolic content (TPC) of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05.
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25 Methanol
80% Alcohol
Hot water
Cold water
g kg–1 RE
20
15
10
5
0
Room
Sun
Oven
Microoven Cross flow
Infra red Freeze dried
Drying method Figure 5. Total flavonoid content (TFC) of Hibiscus sabdariffa leaves subjected to different drying treatments. Values are mean ± SD of three replicates and significant at P < 0.05.
levels of ascorbic acid during food processing is of paramount importance.33 Antioxidant properties Total phenolic content Phenolics are aromatic secondary plant metabolites widely spread throughout the plant kingdom and associated with colour, sensory qualities and nutritional and antioxidant properties of foods. The antioxidant property of phenolics is due to their
redox properties.37 The TPC obtained with different methods of drying and different solvents is shown in Fig. 4. It can be observed that the TPC varied with different solvent extractions and different drying methods. The room-dried leaf cold water extract had a higher TPC content (19.39 g GAE kg−1 DW) than the corresponding organic solvent and hot water extracts, which was further supported by similar observations in Phyllanthus amarus.38 Methanol extracts of fresh samples have a higher TPC than those of processed samples, because methanol can denature
Figure 6. Total antioxidant activity (TAA) of differently dried leaves of Hibiscus sabdariffa. Values are mean ± SD of three replicates and significant at P < 0.05.
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polyphenol oxidases in plant cell wall degradation and thus extract more TPC than water. Not only in leafy vegetables but also in fresh fruits has the superiority of methanol over other solvents for TPC extraction been reported.2,36,39 However, processed plant material extracted with methanol will have lower TPC and antioxidant properties than that extracted with water.38 In dried plant materials, polar compounds are extracted more into polar solvents such as water. It has been proposed that hot water extraction is better than cold water extraction, as there will be a release of cell wall phenolics in the case of hot water extraction.40 However, the order of extractability of TPC in our study was cold water > hot water > 80% alcohol > methanol.
Total antioxidant activity Different sample extracts were used to determine their antioxidant capacities by the formation of phosphomolybdenum complexes. The method is based on the reduction of Mo(VI) to Mo(V) by antioxidant compounds and the formation of a green Mo(V) complex with maximum absorbance at 695 nm. Leaf material of H. sabdariffa dried by different methods exhibited various degrees of antioxidant capacity (Fig. 6). The sun-dried leaf cold water extract showed the highest TAA (69.46 g AAE kg−1 DW), followed by the room-dried leaf 80% alcohol extract (65.01 g AAE kg−1 DW). Variations in antioxidant activity may be due to different phenolic, flavonoid and ascorbic acid contents.
Total flavonoid content Flavonoid compounds are more extractable in organic solvents than in water. The room-dried leaf methanol extract exhibited the highest flavonoid content (19.09 g RE kg−1 DW), followed by the freeze-dried leaf 80% alcohol extract (18.66 g RE kg−1 DW g kg−1 ) (Fig. 5). In addition, the crossflow-dried leaf 80% alcohol extract had a higher flavonoid content (12.12 g RE kg−1 DW) than the room-dried leaf hot and cold water extracts (8.57 and 7.06 g RE kg−1 DW respectively) and the oven-dried leaf hot and cold water extracts (3.21 and 3.13 g RE kg−1 DW respectively). The order of extractability of flavonoids was methanol > 80% alcohol > hot water > cold water.
DPPH free radical-scavenging activity The DPPH free radical-scavenging activity assay is most widely used to determine the primary antioxidant activity of antioxidant compounds in plant and fruit extracts.27 The method is based on the reduction of DPPH radicals in methanol, which causes a drop in absorbance at 517 nm. In this study, ascorbic acid was used as standard antioxidant. The DPPH free radical-scavenging activity of different sample extracts is shown in Fig. 7. All freeze-dried leaf extracts showed higher scavenging activity (cold water 93.7% and hot water 91.7% at 1.25 mg mL−1 and methanol 89.47% and aqueous alcohol 83.47% at 0.625 mg mL−1 ) than ascorbic acid (84.12% at 15 μg mL−1 ). Similarly, the variation in antioxidant
Figure 7. DPPH free radical-scavenging activity of room-dried (RD), sun-dried (SD), oven-dried (OD), microwave-dried (MWD), crossflow-dried (CFD), infrared-dried (IRD) and freeze-dried (FD) leaves of Hibiscus sabdariffa (AA, ascorbic acid standard): a, methanol extracts; b, 80% (v/v) aqueous alcohol extracts; c, hot water extracts; d, cold water extracts. Values are mean ± SD of three replicates and significant at P < 0.05.
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SS Kumar et al.
Figure 8. Ferric-reducing antioxidant power (FRAP) of differently dried leaves of Hibiscus sabdariffa. Values are mean ± SD of three replicates and significant at P < 0.05.
properties of ginger leaves dried by different methods has been studied.4 Although phenols were reported to contribute most towards antioxidant activity, a strong correlation between phenol and pigment contents was also observed.27 Ferric-reducing antioxidant power The ability of the dried leaf extracts to reduce ferric ions was evident and varied with the extraction solvent and drying method used (Fig. 8). The freeze-dried leaf 80% alcohol extract exhibited higher FRAP (35.74 ± 0.87 g AAE kg−1 DW) than the corresponding methanol, cold water and hot water extracts. Iron in ferrous form is common in food systems and its high redox potential contributes to oxidative stress at the cellular level, leading to various physiological dysfunctions.41 The purpose of the test for ferrous ion-chelating activity was to determine the capacity of dried leaf extracts of H. sabdariffa to bind the ferrous ion catalysing oxidation. In this assay the leaf extracts showed chelating activity by capturing ferrous ions before ferrozine. A similar observation was reported for other leafy vegetables.42 Owing to the efficient FRAP of leaf extracts in this study, due to their TPC, TFC and TAA, the postharvest processed leaves of H. sabdariffa can be investigated further systematically to ascertain the level of contribution in protecting cells from metal-induced oxidative stress, with the aim of further supporting H. sabdariffa-fortified food formulations as functional foods. Discussion Overall, different drying methods showed a moderate to low effect on TPC and TFC, indicating that TPC and TFC are rather stable to different dehydration conditions. The low retention of nutrients and antioxidant activity in infrared-, oven- and microwave-dried leaves may have been caused by enzymatic processes, which can lead to substantial changes in the composition of phytochemicals
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and in antioxidant activity.43 Similarly, different drying methods exhibited a profound influence on ascorbic acid content, and all thermal treatments have been reported to significantly decrease the total flavonoid content in soybeans.44 Only 25% of ascorbic acid was retained in sun- and microwave-dried leaf samples, in agreement with similar studies on leaves of Moringa oleifera.5 A number of dehydration procedures such as sun, crossflow, drum, spray, puff, freeze and microwave drying are employed for fruits and vegetables.5 In general, for most vegetables, drying leads to 10–20% loss of pigments such as carotenoids, with the increased surface area of dried or powdered products leading to further losses (through autoxidation) unless they are protected from air and light. Some reports have explained the influence of drying methods on roselle calyxes, wherein the effect of temperature on spray drying of roselle calyx extracts and the retention of monomeric anthocyanins, phenolic compounds, polymeric colour and antioxidant activity were observed upon performing dehydration using fixed bed dryer equipment.11,12 In a recent study the bioactive compound retention potential of aqueous extracts of roselle calyxes and its usage in a dairy beverage product were demonstrated.10
CONCLUSION Room and freeze drying were the best methods for retention of chlorophyll, ascorbic acid and antioxidant compounds of H. sabdariffa leaves. Other methods such as microwave, oven, sun, crossflow and infrared drying had a significant impact on TPC, TFC and TAA. Similarly, water extracts exhibited higher TPC and TAA than organic solvent extracts. The results of this study have implications for H. sabdariffa leaf powder-based processed food formulations in view of its potential quality parameters being retained under suitable drying methods.
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Effect of drying methods on antioxidant potential of roselle foliage
ACKNOWLEDGEMENTS The authors are thankful to the Department of Biotechnology, Government of India, New Delhi for financial assistance. We greatly acknowledge the Director, CSIR-CFTRI for his kind support.
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