Exploring Antioxidant Potential and Quality Attributes of Naturally Sourced Honey from various Geographical regions of India and a Commercial Variant

Sujani Kamble, Tejaswi C*

Department of Pharmacognosy, Government College of Pharmacy,

P. Kalinga Rao Road, Subbaiah Circle, Bengaluru – 560027, Karnataka, India.

*Corresponding Author E-mail: tejutej17@gmail.com

ABSTRACT:

Background and Objectives: Honey is a natural product valued for its nutritional and therapeutic properties, particularly its antioxidant potential. Its quality varies with geographical origin, floral source, and environmental factors, while adulteration remains a challenge. This study aimed to explore and compare the antioxidant activity and physicochemical attributes of honey sourced from diverse geographical regions of India with that of a marketed branded honey, thereby assessing authenticity and quality. Methods: Natural honey samples were collected from five regions of India (Karnataka, Kerala, Himachal Pradesh, Odisha, and Madhya Pradesh) and authenticated through an approved laboratory. Physicochemical parameters (specific gravity, moisture, pH, free acidity, colour, optical density, pollen content, reducing sugars, sucrose, fructose–glucose ratio, hydroxymethylfurfural, viscosity, and electrical conductivity) were determined as per Indian and International Honey Commission standards. Adulteration tests, qualitative, and quantitative estimation of bioactive compounds (total phenolic content [TPC], total flavonoid content [TFC]) were carried out. Antioxidant potential was evaluated using DPPH radical scavenging assay (IC₅₀) and FRAP assay. Correlation and statistical analyses, including Pearson/Spearman correlation were applied. Results: Regional honeys displayed variability in physicochemical traits, bioactive content, and antioxidant activity. TPC showed strong positive correlations with both DPPH IC₅₀ and FRAP, while TFC exhibited a moderate relationship with FRAP. Physicochemical parameters such as colour and optical density were moderately linked with antioxidant indices, whereas hydroxymethylfurfural showed a negative association. Comparative evaluation revealed superior antioxidant activity in several natural honeys relative to the branded sample. Interpretation and Conclusion: Indian honeys demonstrated distinct antioxidant potential and quality profiles depending on their geographical origin. These findings validate their therapeutic value, support honey authentication, and highlight their relevance as functional foods and nutraceuticals.

KEYWORDS: Honey, Antioxidants, Phenolic Compounds, Flavonoids, Free Radical Scavenging Assay, FRAP, Hydroxymethylfurfural, Spectrophotometry, Quality Control.

INTRODUCTION:

Honey is a natural substance produced by honeybees (Apis mellifera and related species) from floral nectars and plant exudates. Beyond its nutritive role, honey has been valued since antiquity for its antimicrobial, anti-inflammatory, and antioxidant activities^1,2. These properties are largely attributed to its complex composition, including sugars, enzymes, organic acids, vitamins, and phytochemicals such as flavonoids and phenolic acids^3,4.

The antioxidant capacity of honey is of particular significance, as oxidative stress is implicated in chronic diseases. Darker honeys, generally richer in phenolics, exhibit stronger free radical scavenging and reducing power^5,6. Importantly, honey quality and bioactivity vary considerably with floral source, geographical origin, and environmental factors. Such variation influences its physicochemical traits and therapeutic value; for example, studies have reported significant differences in antimicrobial efficacy between honeys of different floral origins^7,8.

India, with its diverse flora and climatic conditions, produces a wide range of honeys, but systematic pharmacognostic evaluations remain limited. Comparative studies of antioxidant activity and physicochemical attributes are needed to establish authenticity, detect adulteration, and highlight region-specific strengths^9,10. The present study was therefore designed to compare natural honeys from five Indian states with a commercial variant, focusing on their physicochemical parameters, phenolic and flavonoid content, and antioxidant potential.

MATERIALS AND METHODS:

1. Collection and authentication of samples: Natural honey samples were collected directly from apiaries in five geographically distinct regions of India: Wayanad (Kerala), Bhubaneswar (Odisha), Yellapur (Karnataka), Solan (Himachal Pradesh), and Morena (Madhya Pradesh). A branded commercial honey sample (Dabur, India) was also procured for comparison. Each natural honey was collected in sterile amber containers, sealed to avoid contamination, and transported to the laboratory under controlled conditions. Authentication of the samples was carried out at the Approved Drugs Testing Laboratory, Bengaluru, to confirm compliance with standard definitions of natural honey as per the Bureau of Indian Standards (BIS) and International Honey Commission standards^11-16.

2. Physicochemical evaluation: All physicochemical parameters were evaluated following the Association of Official Analytical Chemists (AOAC, 2016), Bureau of Indian Standards (BIS), and International Honey Commission (IHC) guidelines.

· Specific gravity: Determined using weight per milliliter at 27°C with a calibrated pycnometer.

· Moisture content: Estimated using a refractometric method (Abbe-type ATC refractometer), corrected for temperature.

· pH and Free acidity: pH was measured using a calibrated digital pH meter, while free acidity was titrated with 0.05N NaOH to an endpoint of pH 8.3, expressed in meq/kg.

· Optical density and colour: Optical density was recorded at 660nm and colour intensity at 560nm using a UV–Vis spectrophotometer. Samples were categorized into “white,” “light amber,” “amber,” or “extra light amber” grades.

· Pollen analysis: Pollen grains and plant fragments were quantified microscopically per gram of honey using sediment analysis after centrifugation.

· Sugars: Total reducing sugars, sucrose, and glucose–fructose composition were quantified titrimetrically, and the fructose–glucose ratio was calculated as an authenticity marker.

· Hydroxymethylfurfural (HMF): Measured spectrophotometrically at 284nm and 336nm, calculated as mg/kg.

· Viscosity: Determined using a B-ONE Plus rotational viscometer at 25°C.

· Electrical conductivity: Measured with a calibrated conductivity meter at 20°C.

3. Qualitative phytochemical tests: Preliminary phytochemical screening was conducted using standard procedures: Molisch’s test for carbohydrates, Biuret and Ninhydrin tests for proteins and amino acids, and ferric chloride test for phenolics.

4. Adulteration detection: To ensure authenticity, classical adulteration tests were performed, including the cotton wick test (combustion), Furfural and Fiehe’s test (for cane sugar adulteration), and Aniline chloride test (for added glucose syrup). Results were recorded qualitatively as Pass/Fail.

5. Quantitative estimation of bioactive compounds:

· Total phenolic content (TPC): Estimated using the Folin–Ciocalteu reagent method. Absorbance was measured at 765nm and results expressed as mg gallic acid equivalents (GAE)/g of honey.

· Total flavonoid content (TFC): Quantified using the aluminium chloride colorimetric method, absorbance at 415nm, expressed as mg quercetin equivalents (QE)/g of honey^9,10.

6. Antioxidant assays: Two complementary assays were employed to assess antioxidant activity:

· DPPH radical scavenging assay: 0.1mM DPPH solution in methanol was prepared, and honey extracts were added at varying concentrations. After incubation in the dark for 30minutes, absorbance was measured at 517nm. Radical scavenging activity was expressed as % inhibition, and IC₅₀ values were calculated from dose response curves.

· FRAP (ferric reducing antioxidant power) assay: FRAP reagent (acetate buffer, TPTZ, FeCl₃·6H₂O) was freshly prepared. Honey samples were incubated with the reagent at 37°C for 30minutes, and absorbance recorded at 593 nm. Results were expressed as mg ascorbic acid equivalents (AAE)/g of honey^17,18.

7. Statistical analysis: All experiments were conducted in triplicate, and results were expressed as mean ± standard deviation (SD)^7,8. Statistical analyses were performed using GraphPad Prism (version 9.4.1, GraphPad Software Inc., USA) and Python (version 3.9, with NumPy, pandas, and matplotlib libraries). Pearson’s correlation coefficients (r) were calculated to examine relationships between physicochemical parameters, bioactive compounds, and antioxidant assays. Significance was set at p < 0.05. Correlation patterns were visualized as a heatmap generated using Python, while dose–response curves for DPPH were constructed using GraphPad Prism.^19-21

RESULTS AND DISCUSSION:

Physicochemical parameters: The physicochemical characteristics of honey samples from five geographical regions of India (Wayanad, Bhubaneswar, Yellapur, Solan, and Morena) and one commercial variant are summarized in Table 1.

Moisture content ranged between 16–20%, within the Codex Alimentarius and IHC standards (<20%), confirming good quality and stability⁵^-⁷. Specific gravity values showed an inverse relationship with moisture (r ≈ –0.95), indicating consistency in density–moisture balance^11,12. The pH of samples (3.5–4.5) reflected mild acidity, while free acidity values were below the permissible 50meq/kg limit; their strong negative correlation (r ≈ –0.99) validated the dataset’s chemical reliability^11,12. Electrical conductivity varied by region, being higher in darker honeys, suggesting greater mineral content. Hydroxymethylfurfural (HMF) levels were below 40mg/kg in most samples, except for a slight elevation in one, confirming freshness and minimal heat damage^12,16.

Sugar composition: Reducing sugars (fructose and glucose) constituted 65–75% of total sugars across samples, consistent with BIS and Codex standards. Sucrose content remained <5%, ruling out adulteration. The fructose-to-glucose ratio (1.1–1.4) indicated genuineness and influenced crystallization tendencies.

Table 1: Physicochemical, bioactive, and antioxidant parameters of honey samples collected from five geographical regions of India (Wayanad, Bhubaneswar, Yellapur, Solan, and Morena) compared with a commercial variant. Values are expressed as mean ± SD

Parameters vs Sample	A (Wayanad)	B (Bhubaneshwar)	C (Yellapur)	D (Solan)	E(Morena)	F(Branded)
Specific Gravity (in g/mL)	1.4180 ± 0.01	1.4154 ± 0.01	1.4044 ± 0.01	1.4151 ± 0.01	1.4142 ± 0.01	1.4099 ± 0.01
Moisture Content in %	17.00 ± 0.00	19.00 ± 0.00	21.17 ± 0.29	18.50 ± 0.00	19.00 ± 0.00	20.00 ± 0.00
pH	5.40 ± 0.00	5.75 ± 0.25	6.00 ± 0.10	5.20 ± 0.00	5.20 ± 0.00	5.30 ± 0.00
Free Acidity (meq/kg)	11.14 ± 0.73	8.52 ± 0.57	16.63 ± 0.58	8.09 ± 0.78	11.63 ± 0.58	8.52 ± 1.01
Optical_Density_660 nm	0.347 ± 0.03	0.140 ± 0.00	0.121 ± 0.00	0.105 ± 0.01	0.146 ± 0.00	0.062 ± 0.01
Colour_560 nm	0.762 ± 0.01: Amber	0.372 ± 0.02: Light Amber	0.265 ± 0.01: Light Amber	0.112 ± 0.01: White	0.440 ± 0.01: Light Amber	0.185 ± 0.01: Extra Light Amber
Polen count per gram	18000	14000	15000	12000	12000	13000
Total reducing sugar %	72.168 ± 0.25	76.101 ± 0.00	70.486 ± 0.24	60.601 ± 0.18	76.422 ± 0.28	77.898 ± 0.29
Sucrose %	0.828 ± 0.41	0.612 ± 0.27	0.524 ± 0.23	1.891 ± 0.87	0.461 ± 0.00	0.803 ± 0.28
True Glucose %	34.483 ± 0.35	36.028 ± 0.35	32.757 ± 1.12	27.412 ± 0.35	33.136 ± 3.14	37.221 ± 1.08
True Fructose %	40.740 ± 0.61	43.322 ± 0.38	40.789 ± 1.08	35.880 ± 0.56	46.796 ± 3.10	43.975 ± 0.98
Fructose-glucose ratio	1.182 ± 0.03	1.203 ± 0.02	1.247 ± 0.08	1.309 ± 0.04	1.427 ± 0.24	1.183 ± 0.06
HMF in mg/kg	3.377 ± 0.25	1.657 ± 0.05	2.219 ± 0.35	2.462 ± 0.30	3.685 ± 0.12	3.862 ± 0.12
Viscosity (mPa·s)	1138.33 ± 7.64	1884.00 ± 10.15	1075.00 ± 5.00	1333.33 ± 7.64	1626.00 ± 5.29	1706.67 ± 7.64
Electrical Conductivity, mS/cm	203.33 ± 2.08	491.67 ± 3.51	469.67 ± 8.74	373.33 ± 2.08	424.33 ± 1.53	199.33 ± 1.53
Total Phenolics (in mg GAE/g)	0.620 ± 0.05	0.914 ± 0.03	0.656 ± 0.04	0.342 ± 0.06	0.625 ± 0.04	0.414 ± 0.01
Total Flavonoids (in mg QE/g)	0.098 ± 0.00	0.110 ± 0.00	0.087 ± 0.01	0.040 ± 0.00	0.085 ± 0.01	0.061 ± 0.01
DPPH_IC50	164.7	47.1	467	584.3	73.21	93.23
FRAP (in mg AAE/g)	0.479 ± 0.03	0.871 ± 0.01	0.357 ± 0.03	0.174 ± 0.01	0.738 ± 0.04	0.643 ± 0.05

Hydroxymethylfurfural (HMF) exhibited negative correlations with both antioxidant assays, emphasizing its role as a marker of thermal or storage-related degradation. In contrast, viscosity and electrical conductivity showed weak associations, suggesting limited predictive value for antioxidant potential.

Comparison with Commercial Honey:

The branded honey conformed to physicochemical standards but exhibited lower phenolic and flavonoid content and weaker antioxidant activity than natural samples¹⁸. This is likely attributable to blending, processing, or prolonged storage, which reduce enzymatic activity and bioactive compound stability. In contrast, Bhubaneshwar and Morena honeys retained superior antioxidant strength, reflecting the impact of geographical and floral sources.

Discussion Summary:

Overall, the findings confirm that phenolics are the primary determinants of honey’s antioxidant potential, supported by flavonoids, pigments, and other compositional factors. Natural honeys, particularly from Bhubaneshwar (Odisha) and Morena (Madhya Pradesh), outperformed the commercial variant, underscoring the significance of regional and botanical origin in honey quality. These results align with earlier studies highlighting the influence of polyphenols on honey bioactivity.

The present study highlights the critical role of geographical origin and floral source in determining the quality and antioxidant potential of Indian honeys. Significant variability was observed among the natural samples in terms of phenolic and flavonoid content, physicochemical attributes, and antioxidant capacity. These findings carry multiple implications for both research and practice.

From a food quality perspective, the compliance of all natural samples with Codex Alimentarius and BIS standards confirms their authenticity and consumer safety. Elevated levels of phenolics and flavonoids in Wayanad and Yellapur honeys underscore the contribution of regional biodiversity to honey’s therapeutic properties. Such bioactive rich honeys may serve as superior candidates for functional foods and nutraceutical formulations.

From a consumer and regulatory standpoint, the study reinforces the importance of quality testing and authentication. Adulteration of honey remains a widespread concern in India and globally; therefore, combining physicochemical standards with antioxidant profiling can provide reliable markers for quality assurance. In particular, optical density, colour intensity, and correlation with FRAP and DPPH assays present simple yet powerful indicators that can be used in rapid quality screening.

From a pharmacognosy and therapeutic relevance perspective, the strong antioxidant potential of certain regional honeys supports their traditional use in wound healing, anti-inflammatory therapy, and as immune boosters. These results also align with emerging interest in integrating honey into complementary medicine and dietary interventions for oxidative stress related disorders.

Finally, the comparative analysis with a commercial branded honey highlights the impact of industrial processing and storage on reducing bioactive compound levels. This finding underlines the need for better processing standards and consumer education to preserve the therapeutic value of honey.

Future Scope: The findings of this study provide valuable insights into the quality and antioxidant potential of Indian honeys; however, further work is required to strengthen and expand these observations. Future research may focus on the following aspects:

· Expanded Geographical Sampling: The present study was limited to five representative regions. Inclusion of additional states and seasonal variations would provide a more comprehensive understanding of Indian honey diversity and its nutritional profile.

· Advanced Analytical Techniques: High-performance liquid chromatography (HPLC), mass spectrometry, and nuclear magnetic resonance (NMR) can be employed to profile phenolics, flavonoids, and other phytochemicals in greater detail. This would allow identification of specific bioactive markers linked to antioxidant potential.

· Pollen and Botanical Source Mapping: Melissopalynological analysis combined with DNA barcoding could accurately identify the floral origin of honey samples. Such approaches can strengthen authentication systems and enable labeling of “monofloral” or “multifloral” honeys.

· In Vivo and Clinical Studies: While in vitro assays like DPPH and FRAP provide rapid screening, in vivo models and clinical evaluations are needed to validate the therapeutic potential of antioxidant-rich honeys in managing oxidative stress, inflammation, and related diseases.

· Quality Assurance and Standardization: Incorporating correlation markers such as phenolic content, colour intensity, and antioxidant activity into regulatory frameworks (Codex, FSSAI) would help establish reliable quality standards. This can boost consumer confidence and enhance the global competitiveness of Indian honeys.

· Nutraceutical and Functional Food Development: Richer samples such as Wayanad and Yellapur honeys could be further explored for formulation into capsules, syrups, or fortified dietary supplements, creating value-added products in the nutraceutical sector.

CONCLUSION:

This study demonstrated that honey samples from diverse regions of India differ significantly in their physicochemical properties, bioactive constituents, and antioxidant activity. All natural samples largely complied with international standards, confirming authenticity. Honeys from Bhubaneswar (Odisha) and Morena (Madhya Pradesh) exhibited the highest phenolic and flavonoid content, which strongly correlated with antioxidant potential, while the commercial honey showed comparatively weaker activity. The results highlight phenolics as the primary determinants of antioxidant strength, supported by flavonoids and pigments, and underline the role of color and optical density as rapid quality indicators. These findings reinforce the therapeutic relevance of Indian honeys and emphasize the importance of regional profiling for authenticity, consumer trust, and potential nutraceutical applications.

ACKNOWLEDGEMENTS:

The authors acknowledge the Government College of Pharmacy, Bengaluru, for providing research and laboratory facilities. Guidance of Dr. Sujani Kamble, Assistant Professor, and the valuable support of Dr. Roopashree T. S., Professor and Head of Department of Pharmacognosy, are gratefully acknowledged.

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Received on 19.09.2025 Revised on 11.11.2025

Accepted on 30.12.2025 Published on 21.04.2026

Available online from April 24, 2026

Res. J. Pharmacognosy and Phytochem. 2026; 18(2):117-123.

DOI: 10.52711/0975-4385.2026.00016

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Parameter	DPPH IC₅₀ (r, p)	FRAP (r, p)	Interpretation
Total Phenolic Content (TPC)	r = –0.498, p = 0.315	r ≈ 0.82, p < 0.05	Strong phenolic contribution to reducing power and antioxidant activity.
Total Flavonoid Content (TFC)	r ≈ –0.59, p > 0.05	r ≈ 0.70, p > 0.05	Moderate association with both assays, weaker than TPC.
Optical density / Colour	Negative with IC₅₀	Positive with FRAP	Darker honeys exhibited higher antioxidant potential.
Hydroxymethylfurfural (HMF)	Negative correlation	Negative correlation	Higher HMF linked with reduced antioxidant activity.
Viscosity and Electrical Conductivity	Weak correlations	Weak correlations	Limited predictive value for antioxidant strength.