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COLOR INTENSITY AND ANTIOXIDATIVE CAPACITY OF FOUR NATURAL MONOFLORAL HONEY FROM MALDA, INDIA

Objective: The objective of the study was to explore the color intensity and antioxidative capacity of mango honey available from local niches, in and around Malda, West Bengal, India. 
Methods: Four mango honey samples: LMH1, LMH2, LMH3, and LMH4, were collected from bee hives, in and around Malda, India, and were subjected to determine the color intensity and antioxidative capacity in 2, 2-diphenyl-1-picrylhydrazyl system. 
Results: The honey samples tested had light to dark color variation with color intensity values of 583.59–1842.84 mAU. The total phenolic content (TPC) of the honey samples ranged from 434.52 to 1450.74 μg gallic acid equivalents/ml, while the total flavonoids content (TFC) ranged 39.4–72.67 mg quercetin equivalents/100 g of honey. The honey samples displayed antioxidative activity (AOA) with 9.91–76% inhibition capacity, and the IC50 values of 8.37–19.38 mg/ml. The color intensity of test honeys displayed positive correlation with the TPC (r=0.907) and TFC (r=0.863), while negative correlation between honey color intensity and IC50 values (r=−0.955) was recorded. 
Conclusion: The locally available mango honeys were rich in bioactive components displaying excellent AOA, and thus, the native honey might be utilized as the potential source of natural antioxidant in our part of the globe.


INTRODUCTION
Much attention has been given to consume natural antioxidants, nowadays, as because of the changes in the dietary habit in modern lifestyle [1] and awareness of the unnecessary side effects of the chemical (synthetic) antioxidants. In human body, when the activity of oxidants exceeds the activity of antioxidants and thus antioxidants are favored causing oxidative stress at the cellular levels, which play a role in the pathogenesis and progression of chronic as well as degenerative diseases, such as Alzheimer's disease, atherosclerosis, heart and lung diseases, diabetes, hypertension, and cancer [2,3]. To tackle such a hostile situation, the prime thing is to exogenous administration of antioxidants that comprises micronutrients (Vitamin C and/ or Vitamin E, plant phenolics). Other antioxidant forms (butylated hydroxytoluene; butylated hydroxyanisole; tert-butyl hydroquinone; and propyl gallate), which might otherwise compromise the DNA, protein or (essential) lipid functionalities, were found hazardous to human health [1,4]. However, the natural antioxidants, in dietary materials, have better scavenging properties and are safer and healthier than the synthetic ones [1].
Honey, being a natural medicine, has been in use since ancient times all over the world to combat different infectious diseases as well as non-infectious diseases [10]. The medicinal property and biological activities, including antioxidative capacity, of natural as well as medicated honey, have been reported from various parts of the world [7,[11][12][13]. It has been reported by earlier authors [14,15] that apart from carbohydrates, honey contains compounds such as phenolic acids, ascorbic acid, flavonoids, tocopherols, enzymes (catalase and superoxide dismutase), reduced glutathione, amino acids, and carotenoids; however, the phenolics and flavonoids in honey play a major role as the antioxidants [12,16]. Ahmed et al. [12] studied that the presence of pigments such as carotenoids and flavonoids was related to color of honey, and dark colored honey possesses a large amount of such kind of pigments with higher antioxidation potential. Sohaimy et al. [17] reported that the honey with high color intensity might be the indicative of the presence of a high amount of phenolic compounds and flavonoids in honeys. Earlier the phytochemical components of honey bee products as well as wild honey have been explored mentioning different health benefits, including the antioxidative capacity [18,19]. We also have reported the antibacterial and antioxidative property of two natural honey samples [20]. In the current study, mango honeys from naturally grown bee hives, in and around Malda town, India, were tested to record the antioxidative capacity as well as the color intensity since no scientific data are available on mango honeys from our part of the globe.

Collection of honey samples
Four natural monofloral (mango: Mangifera indica L.) honey samples: LMH1, LMH2, LMH3, and LMH4, were collected, from four different areas (Kendpukur, Sahapur, Chalandar, and Mahinagar, respectively) of Malda district, West Bengal, India, during the month of February, 2017-May 2017. The honey samples were collected by the help of honey collectors, and the identity of the collected honeys was confirmed by the honey collectors depending on the seasonal as well as vegetation variation around the foraging areas of bee hives [15,20]. All the honey samples were kept in the dark at room temperature for further study.

Determination of honey color intensity
The color intensity of honey samples collected was determined following the protocol of Beretta et al. [19]. Briefly, each of the honey samples was diluted to 50% (w/v) with double distilled water (45°C-50°C) and filtered through filter paper (Whatman No.1, Merck), to remove the suspended particles. The absorbance of the honey solution was measured at 450 nm and 720 nm, using double Ultraviolet (UV)-visible spectrophotometer (Lim 332, Labard Instruchem Pvt., Ltd, India), and the difference between the absorbance was considered as the color intensity, and expressed in mAU (milli absorbance units) [21].

Determination of total flavonoids content (TFC)
The TFC was determined using AlCl 3 , according to the protocol of Arvouet-Grand et al. [22] with slight modifications. Briefly, 1.5 ml of 2% AlCl 3 in methanol was mixed with an equal volume of honey solution (1:3, w/v, ratio, in methanol). After incubation at room temperature, for 15 min, in darkness, the absorbance of the mixture was measured at 415 nm against a blank solution (consisting of methanol without AlCl 3 ). The TFC was determined using the calibration curve of quercetin (concentration range: 25-150 µg/ml; R 2 =0.952), and the results were expressed as mg of quercetin equivalents (QE)/100 g of honey.

Antioxidative activity (AOA) of honey
The AOA of each of the honey samples was evaluated in 2, 2-diphenyl-1-picrylhydrazyl (DPPH) system, which was based on the protocol of Habib et al. [23]. The honey solutions: 3-15 mg/ml for LMH1 and LMH2, and 6-30 mg/ml for LMH3 and LMH4, in methanol were prepared, and 0.1 ml of such honey extract (for all honey types of all concentrations) was mixed with 0.3 ml methanol and 0.4 ml 0.3 mM DPPH in methanol, by shaking, and left for 30 min in darkness at room temperature. The reduction (inhibition) of DPPH radical was measured by checking the absorbencies of the mixture at 517 nm, against the control prepared with DPPH in methanol without honey, and the radical scavenging activity (RSA) was calculated as the percentage of DPPH radical inhibition, using the formula: % inhibition =   ControlOD SampleOD 100 ControlOD [24]. The IC 50 (half-maximal inhibitory concentration) value of individual honey samples was determined as per the protocol mentioned earlier [24].

Statistical analysis
The tests were performed in triplicates, and the results have been represented as mean ± standard deviation. The statistical analyses were done using Microsoft Excel 2016. One-way analysis of variance (ANOVA) was used to determine significant differences (p<0.05) among utilized parameters of honey samples. The degree of association between color intensity and other variables (TPC, TFC, and IC 50 ) was assessed by Pearson's correlation coefficient (r).

Color intensity of honey
Among the collected four mango honeys, two (LMH1 and LMH2) were dark amber in color, while the remaining honey samples (LMH3 and LMH4) were light amber in color (Fig. 1). The color intensity test results for honey samples are represented in Table 1 As per the report of Dezmirean et al. [32], the light colored raspberry honey had the lowest amount of total phenolics (148.47 mg GAE/kg), while in the dark colored heather honey the concentration of total phenolics (435.99 mg GAE/kg) was ≈ 3-fold higher than that contained

Antioxidative capacity of honey
The antioxidant property of mango honey samples has been determined in the form of antiradical activity in DPPH scavenging system. The percent inhibition (DPPH reduction/radical scavenging) activities for light color honeys (LMH1 and LMH2) are represented in Fig. 3, and for dark color honeys (LMH3 and LMH4) are represented in Fig. 4

TFC of honey
The quercetin calibration curve in determining TFC is represented in Fig. 2. The TFC of the mango honey tested is shown in

Correlation between honey color intensity and antioxidative profiles
The nature and direction of the correlation between honey color and antioxidative profiles (TPC, TFC, and IC 50 ) are represented in Table 3. An immense association exists between the bio-physicochemical agents (color pattern and color intensity, TPC and TFC) and antioxidative capacity (DPPH RSA) of honey, and the dark color honeys (with high color intensity) possessing higher TFC and TPC demonstrated strong AOA, in the instant study. Dezmirean et al. [32] established a positive correlation between the AOA of honeys and the total phenolics and flavonoids contained in the honeys and showed that the phenolics and flavonoids are responsible for the AOA of honey. As has been reported by Khalil et al. [40], there was a positive correlation between the honey color intensity and various antioxidant parameters of Algerian honeys including TFC and DPPH (0.968 and 0.964, respectively) and between TFC and DPPH RSA (r=0.888). As has been reported by Islam et al. [26], there were considerable correlations between biochemical and antioxidant factors traced in Bangladeshi honeys; the author demonstrated correlation of honey color pattern/color intensity with TPC (r=0.820), TFC (r=0.727), and DPPH activity (r=0.948), and between TPC and DPPH activity (r=0.869) and TFC and DPPH activity (r=0.786). A positive correlation between the presence of phenolic compounds and honey color (r =0.844), the honey color and the antioxidant capacity (r=0.675-0.862), and phenolics and antioxidant capacity (r=0.615-0.782) have been observed, when analyzed in vitro the Brazilian Pampa biome honey, by Cruz et al. [41]. Among six Romanian honeys (black locust, linden, heather, raspberry, canola, and multifloral) tested, the dark colored heather honey contained highest TPC (435.99 mg GAE/kg) as well as TFC (43.17 mg QE/kg) values, while the light colored raspberry honey had TPC and TFC values of 148.47 mg GAE/kg-15.94 mg QE/kg, respectively, with respective AOA (in DPPH system) of 35.77% and 28.9%, for heather honey and raspberry honey [32]. In view of the fact that the honey color reflects, to a certain extent, the content of pigments with antioxidative capacity [42], the current study, for the 1 st time, demonstrated a possible correlation between two such factors: Antioxidative capacity (TFC and TPC) and the color intensity of mango honeys available from local niches.
Moniruzzaman et al. [26] reported the tualang honey among four Malaysian honeys, which had the highest color intensity, highest TPC and TFC value had the highest scavenging activity in DPPH system (59.89%) and concluded that the higher concentrations of phenolic compounds and flavonoids may have been responsible for the higher percentage of radical-scavenging activity. Neupane et al. [43] found a significant correlation between AOA (DPPH RSA) and TPC (r=0.992) of honey from different altitudes of Nepal Himalayas. The TPC in multifloral Poland honeys ranged 611-990 mg/kg, and in Slovak honeys ranged 703.7-1257.12 mg/kg, while in Serbian forest honeys the TPC ranged 769-1052 mg/kg, and the highest TPC (2962.24 mg/kg) was recorded in buckwheat honey, which had highest AOA, by ABTS (4.63 mmol/kg) [44], as well as by DPPH [45]. The honey color, in the current study, showed a high positive correlation with TPC (r=0.907) and TFC (r=0.863) values, and also, the high positive correlations (r=0.988) were achieved between TPC and TFC values of the test honeys, while negative correlations were established between AOA (IC 50 values) and honey color intensity (-0.955), TPC (-0.971), and TFC (-0.967). Thus, the higher the color intensity, the higher the TPC and TFC values of honey and the lower the IC 50 values, the higher the antioxidative activities of the honey.

CONCLUSION
The mango honeys available in local niches are rich in antioxidative components (flavonoids and phenolics), and thus possess excellent antioxidative capacity, wherein the dark colored honey is superior to the light colored honey. The mango honey might be useful as natural antioxidants, and the people in our part of the globe might get beneficial effects on consuming such natural honeys.

AUTHOR'S CONTRIBUTIONS
Surajit Roy performed experimental work and wrote the paper; Shyamapada Mandal designed the study, standardized the protocols, wrote and discussed the paper.