Changes in Antioxidant Activity of Cyphomandra betacea (Cav.) Sendtn. Fruits During Maturation and Senescence

Abstract

Antioxidants are extremely important substances that possess the ability to protect the body from damage caused by free radical induced oxidative stress. Antioxidants are derived from dietary sources, such as fruits, vegetables, and beverages. In this study, the antioxidant activity of different maturity stages of two varieties of Cyphomandra betacea fruits of Darjeeling was evaluated in vitro. The radical scavenging properties on 2,2-diphenyl-1-picrylhydrazyl, superoxide anion, hydroxyl radical, lipid peroxidation, nitric oxide, and reducing power as well as the flavonoids, phenolics, lycopene, and total carotene contents of methanolic extracts of the fruits were determined. All fruit extracts, mainly the mature red fruit of purple-red variety exhibited strong scavenging activity towards all radicals tested due to the presence of relatively high total phenol, flavonoids, and lycopene as well as total carotene contents. The findings suggest that purple red variety of C. betacea fruit is endowed with antioxidant phytochemicals, which could provide protection against oxidative stress induced diseases.

Keywords:

• Cyphomandra betacea
• Antioxidant
• DPPH
• Superoxide
• Phytochemicals

INTRODUCTION

Oxidative stress is the major causal factor for the induction of many chronic and degenerative diseases, including atherosclerosis, ischemic heart disease, aging, diabetes mellitus, malignancy, immunosuppression, neurodegenerative diseases, and others.[1–6 6 Free radicals, which are the major sources of oxidative stress, can only be removed by antioxidants of natural and synthetic origin.[7–9 9 In recent times, natural antioxidants have attracted considerable interest among nutritionists, food manufacturers, and consumers, due to their presumed safety and potential therapeutic values. Plants contain a wide variety of free radical scavenging molecules, such as phenols, flavonoids, vitamins, and terpenoids, that are rich in antioxidant activity.[10] Natural phytochemicals with potential antioxidant values are found in crops, beverages, oilseeds, beans, fruits, and vegetables.[11] Several herbs and spices have been reported to exhibit antioxidant activity, including rosemary, sage, thyme, nutmeg, turmeric, white pepper, chili pepper, ginger, and several Chinese medicinal plants.[12–14 14

Cyphomandra betacea (Cav.) Sendtn. is a commonly grown plant in the hills of Darjeeling, and the fruit is popularly known among Nepalese as Rukh rambera or Rukh tamatar. The fruits are eaten fresh, cooked in stews and sauces, prepared as chutney and pickles, as well as directly consumed with salads. This fruit, typically acidic, is recommended for its nutritional qualities as a good source of provitamin A, vitamins C, B₆, and E, and iron.[15–18 18 According to Tene et al.,[19] C. betacea mature fruit juice is traditionally used in Ecuador for curing tonsillitis, high cholesterol, and stomach pain.

The aim of the present study was to investigate the fruits of golden yellow and purple-red varieties of C. betacea from Darjeeling hills as a potential functional food and antioxidant source, as an alternative to synthetic compounds. In this article, we have determined the radical scavenging efficacy of fruits during their different stages of maturation as well as the phytonutrients of these fruits, such as total carotene, lycopene, total phenolics, and flavonol.

MATERIALS AND METHODS

Plant Samples

Golden-yellow (Fig. 1) and purple-red (Fig. 2) variety of C. betacea fruits were collected on February 16, 2009 at different stages of their maturity from Takdha Basti, Darjeeling, West Bengal, India. Taxonomic position was authenticated by the Taxonomy and Environmental Biology Laboratory, Department of Botany, University of North Bengal. The material has been deposited in the ‘NBU Herbarium’ and recorded against the accession number 9579, dated 04-03-09.

Figure 2 Different stages of maturity of Purple-Red variety of C. betacea fruit. (Color figure available online.)

Figure 1 Different stages of maturity of Golden-Yellow variety of C. betacea fruit. (Color figure available online.)

Preparation of Fruit Extracts

The fruits of two varieties of C. betacea at three different mature stages were cut into small pieces and were separately crushed with mortar and pestle. Under a Soxhlet extractor, crushed fruits were separately extracted with methanol water in a ratio of 4:1 (v/v) for 8 h. The supernatants of refluxed samples were isolated from the residues by filtering through Whatman No. 1 filter paper. The filtrates were dried in vacuo by rotary evaporator and their total extractive values were calculated on dry weight basis by the formula:

The samples were then kept in a freezer for further use.

Animal Material

Goat liver, used for anti-lipid peroxidation assay, was collected from a slaughterhouse immediately after slaying and the experiment was conducted within 1 h after collection.

Chemicals

Methanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ethylene diamine tetra acetic acid sodium salt (Na-EDTA), ascorbic acid, 2-deoxyribose, ferric chloride (FeCl₃), hydrogen peroxide(H₂O₂), hydrated ferrous sulphate (FeSO₄.7H₂O), thiobarbituric acid (TBA), trichloroacetic acid (TCA), sodium nitroprusside, nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide phosphate reduced (NADPH), phenazine methosulphate (PMS), Folin-ciocalteu reagent, sodium carbonate (NaCO₃), sodium nitrite (NaNO₂), aluminium chloride (AlCl₃), Griess reagent, petroleum ether, acetone and sodium sulphate (Na₂SO₄) were either purchased from Himedia-BDH (Mumbai, India) or Merck (Darmstadt, Germany). All the chemicals were of analytical grade.

Determination of DPPH Radical Scavenging Assay

Radical scavenging activity of plant extracts against stable DPPH (2,2-diphenyl-1-picrylhydrazyl) was determined spectrophotometrically. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in color (from deep-violet to light-yellow) were measured at 517 nm wavelength. Radical scavenging activity of extracts was measured by a standard method.[20] Two microliters of each sample, prepared at various concentrations (10, 20, 50, 100, 250 mg/ml), were added to 2 ml of 0.2 mM DPPH solution. The mixture was shaken and allowed to stand for 30 min at 20°C, and then the absorbance was measured at 517 nm with UV-VIS spectrophotometer (Model No. 2201, Systronics, Ahmedabad, India). The percentage of inhibition activity was calculated by the following equation:

where A _control is the initial concentration of the stable DPPH radical without the test compound and A _sample is the absorbance of the remaining concentration of DPPH in the presence of methanol. IC₅₀ values (mg/ml) were determined from a plotted graph of scavenging activity against the concentrations of the C. betacea fruit extracts, where IC₅₀ is defined as the total amount of antioxidant necessary to decrease the initial DPPH radical concentration by 50%.

Determination of Superoxide Anions Scavenging Activity

The superoxide anions generated by phenazine methosulphate (PMN) and nicotinamide-adanine dinucleotidphosphate, reduced form (NADPH), were detected by the reaction with 2,2′-di-p-nitrophenyl-5,5′-diphenyl-(3,3′-dimethoxy-4,4′-diphenylene) di-tetrazolium chloride (nitro blue tetrazolium-NBT).[21] Reaction mixture contained 1 ml samples (different concentration), 1 ml of NBT solution (312 μM prepared in phosphate buffer, pH 7.4) and 1 ml of NADH solution (936 μM prepared in phosphate buffer, pH 7.4). Finally, the reaction was accelerated by adding 100 μL of PMS solution (120 μM prepared in phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25°C for 5 min and absorbance at 560 nm was measured against methanol as the control. Percentage inhibition and IC₅₀ value was calculated using the same formula mentioned above.

Determination of Reducing Power

One milliliter of fruit extract, 2.5 ml of sodium phosphate buffer (0.2 M, pH 6.6), and 2.5 ml of potassium ferricyanide (1% w/v) were incubated at 50°C for 20 min. The tube was cooled on ice and 2.5 ml of 10% trichloroacetic acid was added. The mixture was centrifuged at 3000 rpm for 10 min to collect the upper layer of solution (2.5 ml) and mixed with distilled water (2.5 ml) and 0.25 ml of FeCl₃ (0.1% w/v). Finally, the absorbance was measured at 700 nm against a blank sample.[22]

Determination of Hydroxyl Radical Scavenging Activity

Hydroxyl radical scavenging activity was determined by using the 2-deoxyribose oxidation assay.[23] A solution (0.2 ml) of 10 mM FeSO₄, 7H₂O, and 10 mM ethylenediamine tetraacetic acid was prepared in a screw capped test tube. Then, 0.2 ml of 10 mM 2-deoxyribose solution, 0.5 ml of each sample (different concentration), and 0.1 M sodium phosphate buffer (pH 7.4) were added to give a total volume of 1.8 ml. Finally, 200 μl of 10 mM H₂O₂ solution were added to this reaction mixture and incubated at 37°C for 120 min. After incubation, 1 ml each of 2.8% trichoroacetic acid and 1.0% thiobarbituric acid were added to the reaction mixture. The sample was boiled at 100°C for 10 min, cooled in ice, and then its absorbance was measured with a spectrophotometer at 515 nm. The IC₅₀ value was calculated by the same process mentioned above.

Determination of Nitric Oxide Activity

Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reaction.[24] First, 320 μL of methanol extract, 360 μL (5 mM) of sodium nitroprusside-PBS solution, 216 μL of Greiss reagent (1% sulfanilamide, 2% H₃PO₄, and 0.1% napthylethylenediamine dihydrochloride) was mixed and incubated at 25°C for 1 h. Finally, 2 ml of water were added and absorbance was taken at 546 nm. The IC₅₀ value was calculated by the same procedure mentioned above.

Anti-Lipid Peroxidation (ALP) Assay

The anti-lipid peroxidation activity of the extracts of fruits was determined by the standard method followed by a slight modification with the goat liver homogenate.[25] First, 2.8 ml of 10% goat liver homogenate, 0.1 ml of 50 mM FeSO₄, and 0.1 ml extract were mixed. This mixture was incubated for 30 min at 37°C. Then 1 ml of the reaction mixture was taken with 2 ml of 10% TCA–0.67% TBA in acetic acid (50%) for blocking the reaction. The mixture was then boiled for 1 h at 100°C and centrifuged at 10,000 rpm for 5 min. Supernatant was taken for absorbance at 535 nm. Butylated hydroxytoluene was used for standard. ALP % was calculated by using the following formula:

Total Phenol Estimation

Total phenolic compounds of fruit extracts were determined by Folin-Ciocalteu method.[26] For the preparation of the calibration curve, 1 ml aliquot of 0.025, 0.05, 0.075, 0.1, 0.2, and 0.3 mg/ml methanolic gallic acid solution was mixed with 5 ml of Folin-Ciocalteu reagent (10 times diluted) and 4 ml of sodium carbonate (75 g/L). The absorbance at 765 nm was measured after 1 h at 20°C and the calibration curve was drawn. Then 1 ml of methanolic fruit extracts (50 mg/ml fresh weight tissue) was mixed to the same reagent and the mixture was incubated for 1 h in room temperature. After 1 h, the absorbance was measured at 765 nm.

Total Flavonoids Determination

A spectrophotometric aluminum chloride method was used for flavonoid determination.[27] Each of the fruit methanol extracts (0.5 ml of 100 mg/ml FW) were separately diluted with 4 ml of double-distilled water. Then the diluted fruit extracts were mixed with 5% (0.3 ml) NaNO₂ and 10% aluminum chloride was then added with the reaction mixture. After 6 min, 2 ml (1.0 M) NaOH and 2.4 ml double distilled water were added and mixed well. Thereafter, absorbance was measured at 510 nm by a spectrophotometer. Standard solution of quercetine (0–500 mg L⁻¹) was used as the calibration curve.

Estimation of Lycopene Content

Lycopene content was measured spectrophotometrically at one of its absorption maxima.[28] First, 5 ml of methanolic fruit extract was dissolved with 15 ml of acetone. The extract was then transferred to a separating funnel containing about 20 ml of petroleum ether and mixed adequately. To it, about 20 ml of 5% Na₂SO₄ solution was added and gently shaken. Next, 20 ml of petroleum ether was added again to the funnel for clear separation of the two layers. The upper layer was mostly colored. The two phases were separated and the lower aqueous phase was re-extracted with 20 ml of petroleum ether until the aqueous phase become colorless. The petroleum ether extract was pooled and washed once with a small amount of distilled water. The petroleum ether extracts enriched with lycopene were transferred into a brown bottle containing about 10 gm of anhydrous Na₂SO₄ and kept aside for 30 min. Petroleum ether extract was decanted into a 100-ml volumetric flask through a funnel containing cotton wool. The Na₂SO₄ slurry was washed with petroleum ether until it became colorless and was transferred to the volumetric flask. Finally, the volume was made up and the absorbance was measured in a spectrophotometer at 503 nm using petroleum ether as the blank. The lycopene content was calculated by the formula:

where 1 O.D = 3.1206 μg/gm.

Estimation of the Total Carotene Content

Total carotenoids were estimated spectrophotometrically through solubility-based solvent partitioning followed by hydrolysis of bound carotenoid esters with concentrated KOH solution.[28] First, 1 ml of methanolic fruit extract was mixed with 2 ml of petroleum ether in a separating funnel. Two layers of aqueous phase were observed. The upper phase was collected, whereas the lower phase was decanted; this procedure was repeated thrice. The collected upper phase containing the carotenoids was evaporated at 37°C and the residue was dissolved in 2 ml of ethanol. Next, 2 ml of aqueous 60% KOH was added to the residue mixture and boiled for 5 to 10 min. An equal volume of water (i.e., 2 ml) was added and partitioned thrice with petroleum ether and evaporated. To the reaction mixture, 2.5 ml of ethanol was added and the absorbance was taken at 450 nm. The carotene content was calculated by the formula:

where

	D = Absorbance value;
	V = Volume of original extract in ml;
	F = Dilution factor;
	2500 = Average extinction coefficient of the pigment.

Statistical Analysis

The data were pooled in triplicate and subjected to analysis of correlation co-efficient matrix using SPSS (Version 12.00, SPSS Inc., Chicago, IL, USA) for drawing the relation between phytochemicals and antioxidant attributes and MS Excel 2007 (Microsoft, Redmond, WA, USA) was used for comparing the antioxidant attributes of different maturation stages of the C. betacea fruits. The data were analyzed by one-way ANOVA and different group means were compared by Duncan's Multiple Range Test (DMRT) through DSAASTAT software (version 1.002; DSAASTAT, Peruglia, Italy); p < 0.05 was considered significant in all cases. The software package Statistica (Statsoft Inc., Tulsa, OK, USA) was used for analysis of other data. Smith's Statistical Package version 2.5 (prepared by Gary Smith, CA, USA) was used for determining the IC₅₀ values of antioxidants and their standard error of estimates (SEE).

RESULTS AND DISCUSSION

According to Vasco et al.[29] the peels of both purple-red and golden-yellow variety and the seed-jelly of the purple-red C. betacea have high absorbance at 520 nm due to their anthocyanin content. These authors classified C. betacea fruits as a low antiradical efficiency drug against the DPPH free radical, when compared with other fruits available in Ecuador, but our study revealed that the methanolic extract of C. betacea exhibited strong DPPH radical scavenging activity, which is in agreement with the views of Kou et al.[30] In the previous work by Ordonez et al.,[31] the DPPH scavenging activity of C. betacea fruit and their macerated products were similar to or higher than that of natural antioxidants like ascorbic acid. Anthocyanins are one of the important groups of compounds associated with antioxidant activity. Synthetic free radical DPPH can be efficiently used for determining antioxidant activity of several natural defense molecules like cysteine, glutathione, ascorbic acid, tocopherol, and other poly-hydroxy aromatic phenylpropanoids.[20] Table 1 shows that DPPH free-radical scavenging efficacy is significantly higher in the purple-red variety than in the golden-yellow variety. In both varieties, the IC₅₀ values are gradually decreased towards immature to mature fruits, which indicate that the accumulation of antioxidant compounds are associated with ripening. Similar observations were also recorded by Vasco et al.,[29] when the seed jelly, pulp, and peel of both of the varieties of C. betacea were compared. According to Heatherbell et al.,[32] marked increase in anthocyanin and organic acids were observed during maturation. These compounds may have a structure function relationship with antioxidants or both were strengthened during development. Mature fruits of other plants, like mulberry, also exhibited similar properties as claimed by Wang and Hu,[33] who concluded that only mature stages and their interaction with their genotype were significant for DPPH scavenging activity. Methanol extractive values were also exhibited in a similar pattern as revealed from Table 1, and were attained optimally during fruit senescence of both varieties.

Carotenoid biosynthesis and its regulation during development and ripening of fruits of Solanaceae is a complex process that occurs during transformation from chloroplasts into chromoplasts and associated changes of the organoleptic properties of the fruit.[34] Table 1 explains that total carotene content is present in high quantity in the mature stages of both varieties (3.42 and 3.48 mg per kg in golden-yellow and purple-red variety, respectively). Our study revealed that the total carotenoids of golden-yellow and purple-red fruits of C. betacea were comparable, which is in contrast with the findings of Vasco et al.,[35] who claimed that the purple-red variety had higher levels of β-carotene. Total carotenoids of C. betacea were superior to those of most tropical fruits, making them functionally interesting. But the variability of results that were evidenced by different authors may be due to the differences in polarity of the solvents chosen and quantification techniques.[36] Of all the carotenoid pigments, lycopene is the most efficient singlet oxygen quencher.[37] cis-Lycopene isomers were found to have higher antioxidant potential with an estimate of twice the activity of all-trans-α-carotene.[37] The ripe tomato accumulates a large amount of lycopene, which is also revealed from the pattern of gene expression found during fruit ripening.[29] The purple-red variety of C. betacea contains the highest amount (0.406 μg/g) of lycopene in mature red fruits (Table 1).

Table 1  Functional components and antioxidant properties of Cyphomandra betacea (cv. Golden Yellow and Purple Red) at different stages of maturity.^a

	Golden Yellow			Purple Red
Functional component/property (unit)^b	Green	Yellow	Orange	Green	Purple	Red
Carotene (mg/100g)	0.214 ± 0.016b	0.252 ± 0.012b	0.348 ± 0.012a	0.214 ± 0.016b	0.204 ± 0.011b	0.342 ± 0.048a
Lycopene (μg/g)	0.070 ± 0.008d	0.094 ± 0.014cd	0.100 ± 0.014cd	0.119 ± 0.010bc	0.156 ± 0.018b	0.406 ± 0.032a
Phenol content (mg/g)	0.612 ± 0.046a	0.787 ± 0.036ab	0.864 ± 0.031ab	0.618 ± 0.049a	0.750 ± 0.040ab	1.148 ± 0.086b
Flavonol content (mg/g)	0.106 ± 0.022a	0.199 ± 0.030b	0.308 ± 0.033c	0.116 ± 0.031a	0.215 ± 0.030b	0.401 ± 0.086d
DPPH (mg/ml)	98.497 ± 10.560c	89.250 ± 7.810bc	82.021 ± 7.240bc	92.497 ± 9.480c	71.140 ± 6.820ab	61.440 ± 4.360a
Methanol extractive values (%)	2.770 ± 0.520a	6.430 ± 0.340c	6.650 ± 0.670c	2.500 ± 0.440a	5.850 ± 0.560c	4.600 ± 0.820b
Superoxide scavenging activity (mg/ml)	323.610 ± 35.414e	194.417 ± 17.013d	69.036 ± 6.094b	290.000 ± 29.759e	117.433 ± 11.258c	11.978 ± 2.850a
Hydroxyl radical (mg/ml)	1019.110 ± 111.525b	859.090 ± 75.176ab	701.800 ± 61.948a	953.900 ± 97.887b	740.540 ± 70.994a	682.700 ± 48.447a
NO (mg/ml)	4158.018 ± 455.026c	3687.333 ± 322.667c	2688.492 ± 237.313b	3876.000 ± 397.745c	2616.366 ± 250.824b	1870.577 ± 132.743a
Inhibition of lipid peroxidation (%)	25.424 ± 2.915a	59.217 ± 5.182b	62.439 ± 5.512b	31.641 ± 2.734a	69.444 ± 6.657bc	74.495 ± 5.286c
Reducing power (absorbance at 700 nm)	0.382 ± 0.029d	0.582 ± 0.027c	0.752 ± 0.057b	0.391 ± 0.069d	0.471 ± 0.035cd	1.103 ± 0.118a
^aResults are expressed as Mean ± SEM of triplicate determinations. Values with different letters (a, b, c, etc.) differ significantly (p ≤ 0.05) by Duncan's Multiple Range Test (DMRT).
^bOn fresh-weight basis.

Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids.[38] Phenolic acids have been implicated as natural antioxidants in fruits, vegetables, and other plants. For example, caffeic acid, ferulic acid, and vanillic acid are widely distributed in the plant kingdom.[7] The total soluble phenolic contents for golden-yellow and purple-red C. betacea are presented in Table 1. Higher amounts of total phenols and flavonols were present in the purple-red than in the golden-yellow fruit variety (Table 1). This is in agreement with the findings of Vasco et al.,[29] who analyzed total soluble phenolic compounds and flavonols separately in the peel, pulp, seed jelly, and whole fruits of both varieties. The data available in Table 1 demonstrated that the phenolic components and flavonoids increased steadily with maturation. Other authors also inferred that ripe fruits of C. betacea were the potential source of free phenolics, flavones, flavonone, anthocyanins, and other phenylpropanoid derivatives.[31]

A wide variety of naturally occurring phenyl propanoid derivatives, including flavonoids, isoflavones, flavones, and catechins, can prevent or reduce oxidative stress by scavenging free radicals.[9,39] During oxidative stress, large quantities of reactive oxygen species (ROS) like hydrogen peroxide, superoxide, hydroxyl radical, singlet oxygen, and nitrogen species are generated. These ROS have a role in degenerative disease and early apoptosis in animals.[40] Although the superoxide anions are relatively weak oxidants, it can combine with nitric oxide, to give more reactive species.[41,42] In this study, the superoxide anion scavenging effects of two maturation stages of C. betacea were analyzed by the PMS-NADH superoxide generating system. Methanolic extracts of this fruit, mainly a mature red one, exhibited outstanding superoxide anion radical scavenging activity (IC₅₀ value 11.98 mg/ml) (Table 1).

Among the oxygen radicals, the hydroxyl radical, nitric oxide, and lipid peroxidation induce severe damage to adjacent bio-molecules in cells causing cell death. Thus, the removal of these radicals are very important for the protection of the living system.[43] Like superoxide, the scavenging activities against hydroxyl, nitric oxide, and lipid peroxidation were optimal in the mature red fruit of the purple-red variety of C. betacea. The antioxidant activities were enhanced successively during immature to mature transition (Table 1). Unlike others, the scavenging effect against nitric oxide was not so impressive (Table 1).

In the reducing power assay, the presence of antioxidant in the C. betacea fruit extracts catalyze the reduction of oxidized Fe³⁺/ferricyanide complex to ferrous (Fe²⁺) form. Therefore, the amount of Fe²⁺ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm. Enhancing absorbance indicates an increase in reducing ability. From the results represented in Table 1, it may be concluded that the optimized reducing potential of fruit extracts of two mature varieties was obtained at a concentration of 50 mg/ml (FWT).

Table 2  Correlation matrix

	DPPH	SO	RP	EV	OH	NO	ALP	TFC	TPC	CARO
SO	0.907(*)
RP	−0.763	−0.869(*)
EV	−0.449	−0.672	0.417
OH	0.881(*)	0.987(**)	−0.786	−0.723
NO	0.966(**)	0.966(**)	−0.828(*)	−0.492	0.949(**)
ALP	−0.889(*)	−0.936(**)	0.734	0.800	−0.937(**)	−0.879(*)
TFC	−0.868(*)	−0.963(**)	0.968(**)	0.550	−0.910(*)	−0.929(**)	0.857(*)
TPC	−0.857(*)	−0.898(*)	0.981(**)	0.434	−0.819(*)	−0.880(*)	0.809	0.972(**)
CARO	−0.528	−0.774	0.916(*)	0.454	−0.711	−0.678	0.577	0.875(*)	0.826(*)
LYCO	−0.831(*)	−0.685	0.796	0.005	−0.597	−0.792	0.581	0.769	0.862(*)	0.525
DPPH: 2,2-diphenyl-1-picryl hydrazyl scavenging activity; SO: superoxide radical scavenging activity; RP: ferric reducing antioxidant power; EV: extractive value; OH: hydroxyl radical scavenging activity; NO: nitric oxide scavenging activity; ALP: anti-lipid peroxidation activity; TFC: total flavonol content; TPC: total phenol content; CARO: total carotene content; LYCO: total lycopene content.
Figures in bold indicate significant correlation; (*,**) = significant at p < 0.05 or 0.01, respectively.

The correlation between total phenol content and antioxidant activity has been widely studied. Some reports suggested a high linear correlation between phenolic components and radical scavenging activity in different fruits and vegetables.[44–46 46 In the present study, significant correlations were obtained between free-radical scavenging activity and total phenol content of the sample. As prescribed in Table 2, total phenol is genuinely correlated with DPPH, superoxide, hydroxyl, and nitric oxide radicals along with reducing power of the samples. In the case of flavonoids, wide significant correlations were observed. These observations are in agreement with Ordonez, who indicated that the phenolic compounds and the flavonoids contributed to antioxidant activity more than anthocyanin.[31] However, good correlations were not established between free-radical scavenging and total carotenoids of C. betacea (Table 2). Interestingly, a superior correlation was registered between lipophilic lycopene content, which is a specific type of carotenoid pigment, and partially hydrophilic DPPH radical scavenging (r ² = −0.831) but poorly correlated with other radical scavengers, which are purely hydrophilic in nature. Some authors reported that the concentration of chlorogenic acid in the fruit like tomato decreases during maturation induced ripening with simultaneous increase of ferulic and caffeic acids along with the augmentation of lycopene at the same time.[37, 47] This interpretation is supported by the positive correlations found between total phenol content and lycopene (r ² = 0.862 at p < 0.05) and between total phenol/flavonol content and carotenoids (r ² = 0.875/0.826 at p < 0.05) (Table 2). Recent research demonstrated that phenolic rich ethyl acetate fraction inhibited TBARS formation and scavenge DPPH and ABTS⁺ radical more effectively than n-butanol and aqueous fractions of C. betacea.[30] These authors also concluded that C. betacea phenolics are potent antioxidants, which can inhibit LDL oxidation in vitro and ROS production in a rat adrenal pheochromocytoma cell line. HPLC analysis of golden-yellow and purple-red variety of C. betacea confirmed the existence of hydrocinnamic acid derivatives among free phenolics and the flavonol glycosides, like quercetin and myricetin, were also present in a significant amount.[29] Whatever may be the fact, C. betacea fruits are an important source of antioxidants mainly contributed by a diverse group of phenyl propanoid derivatives as established by various authors from different perspectives. When the edibility is concerned, the fruit is quite safe for consumption as the extracts of C. betacea fruits and their different preparations did not exert any toxic or mutagenic effect in standard strains of Salmonella typhimurium cultured in vitro[31] or in different organs of rat when administered in vivo.[48]

CONCLUSION

The present study suggested that methanolic fruit extract of Cyphomandra betacea are rich in antioxidants and are an important source of lycopene. They are also an abundant source of total carotene, flavonol, and phenolic compounds, which might be helpful in preventing or slowing the progress of various oxidative stress-related diseases. From our results, it may be concluded that the free radical scavenging activities of these fruits increased gradually during maturation. Further investigation on isolation and identification of antioxidant components of this plant may lead to chemical entities with a potential for clinical use.

ACKNOWLEDGMENT

The authors are grateful to the University Grants Commission (UGC) for supporting this research under Meritorious Scheme.