Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 3 2 - 3 9 Research article DOI: https://doi.org/10.54796/njb.v10i1.228 32 ©NJB, BSN 32 Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal Abhishek Bajgain, Bashu Dev Neupane, Diwakar Sarraf, Jwalant Karmacharya, Saksham Ranjitkar, Rajan Shrestha, Rajendra Gyawali Department of Pharmacy, School of Science, Kathmandu University, Dhulikhel, Kavre, Nepal Received: 9 Jun 2022; Revised: 22 Jun 2022; Accepted: 4 Jul 2022; Published online: 30 Jul 2022 Abstract Honey is a natural sweet substance produced by Apis sp. from floral nectar or other plant parts which are gathered, modified and stored in the honeycombs by honeybees. The current research was aimed to analyze the quality parameters of locally available honey. Honey samples of Apis laboriosa and Apis mellifera were collected during spring of 2019 & 2021 and autumn 2021 from the Bagmati province, Nepal. Samples were analyzed their physicochemical and phytochemical properties. The result shows that, the pH was ranged between [4.467±0.0306 - 5.05±0.02], rheological studies showed Newtonian flow and pseudo- plastic type of Non-Newtonian flow, specific optical rotation was ranged between [(+) 5.75±0.4684 - (-) 12.71±0.234], specific gravity was ranged between [1.35±0.00017 - 1.409±0.00022], moisture content was ranged between [19.2% - 25%]. Secondary Metabolite screening showed the honey samples possesses flavonoids, saponins, glycosides, tannins, amino acids, protein and reducing sugar. Total phenolic content was ranged between [1.0427 - 6.86288] gm GAE/Kg honey while total flavonoid content ranged between [0.016755 - 0.353132] gm QE/Kg Honey. IC50 obtained from DPPH assay ranged between [649.6465 - 9867.1617] ppm. Properties and qualities of honey are affected by seasonal factors and various floral sources. The samples were in positive correlation between flavanoid content, phenolic content and their respective anti-oxidant potency. Keywords: Honey, Quality, Physicochemical Properties, Phytochemicals, TPC, TFC, Antioxidant. Corresponding author, email: ragyawali@gmail.com Introduction Honey is a natural sweet substance produced by honey bee, which can be classified based on type of honey source, floral and extra floral honey[1]. Apis laboriosa is world’s largest honeybee species with measurement of up to 3.0 cm, which are found at an altitude range from 2,500 m to 4,000 m above the sea level, building their nests commonly in higher altitude, 1,200 m. above the sea level. Honey is harvested twice a year in spring season and in autumn season, with Spring one showing strong medicinal property than autumn one [2].The medicinal property of honey is that, the floral distribution of the region, where Apis laboriosa lives; is the plants that belong to Ericaceae (Rhododendron) family, which have a psychoactive and hallucinogenic group of phytochemistry known as Grayanotoxins[3]. In Nepal, it is estimated that over 10,000 MT of honey is produced and every year growing the production. Honey is reported to contain at least 181 substances, high nutritional value, high refractive index, high viscosity and Specific gravity [4][5][6]. To the best our knowledge, this is the first comparative study on Nepalese honey to investigate wide range of quality parameters. The present study was aimed to carry out the quality assessment and characterization of honey of Apis laboriosa harvested from Bagmati province, Nepal. Material and methods Honey Collection In this study, honey samples of Apis laboriosa and Apis mellifera were harvested on spring of 2019 & 2021 and autumn 2021 from Bagmati Province, Nepal. The information about samples profile is as given below in the Figure 1, Figure 2 and Table 1. Physical Properties In a view to identifying the purity of honey based on physical properties following preliminary tests were performed. Sand Sinking Test In this test, 50 g sand was taken from nearby and was sieved through Mesh Size 30. The sand was then allowed to dry completely in oven at a temperature of 95℃ for 15 Minutes. The particle distribution profile of the sand was examined using electromagnetic sieve shaker (Model: EMS-8, Instrument Manufacturer: Electropharma). After that, four petri plates were kept in a row. Nepal Journal of Biotechnology Publisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 Journal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 https://orcid.org/0000-0002-5745-0702 mailto:ragyawali@gmail.com Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):3 2 - 3 9 Bajgain et al. 33 ©NJB, BSN 33 Figure 1. Google Earth Snapshot showing Honey Harvested Location Figure 2.f Different honey samples (From Left to Right; S1, S2, S3, and S4). With the help of a tripod stand, the funnel was set and the sand was allowed to flow through the funnel, which created a conical shape of sand over the petriplates. To the sandhill, three drops of each honey sample were placed in each petriplates, and the time required to sink through the sand surface was recorded. Delayed sinking Honey samples are considered to be pure as those honey samples contain less amount of moisture and are not adulterated [7]. Water Sinking Test In this test, a 5 g of each sample was taken and it was poured into a beaker containing deionized water. After that, the nature by which it interacts with water was observed. Directly sinking honey samples to the bottom of the vessel without mixing with water unless stirred are considered to be pure [7]. Air-Flow Test In this test, a clean glass stirrer was taken and was dipped into the vessel containing the honey sample. The stirrer was then rotated through the honey sample Table 1. Honey collection site of Bagmati Province, Nepal Sample No. Harvested Time Types of Honey Honeybee Species Harvested Location Year Season S1 2019 Spring Honeydew Honey Apis laboriosa Uttargaya R.M. – 1, Karyangmaryang, Rasuwa [39.1 Km Perimeter, 71.9 sq. Km Area] S2 2021 Spring General Nectar Honey Apis laboriosa Uttargaya R.M. – 2, Thulogaun, Rasuwa [28° 1'28.36"N, 85° 9'50.73"E] S3 2021 Autumn General Nectar Honey Apis laboriosa Uttargaya R.M. – 2, Thulogaun, Rasuwa [28° 1'28.36"N, 85° 9'50.73"E] S4 2021 Autumn Diploknema butyraceae Nectar Honey Apis mellifera Rakshirang R.M., Silinge, Makwanpur [42.7 Km Perimeter, 48.8 sq. Km Area] Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):3 2 - 3 9 Bajgain et al. 34 ©NJB, BSN 34 and it was raised to a certain height above the honey surface to allow for free-flow of the honey samples. The flowing nature of honey was then observed. Honey samples, which flow in a continuous thread-like pattern, are considered to be pure [7]. Physicochemical Properties Colour Colour tone of honey was noted, which also revealed the identity and nature of various floral sources and helped in the differentiation of types of honey. pH Firstly, the pH meter (Model: pH 211 microprocessor, instrument manufacturer: Hanna Instruments) was calibrated using Qualigen (Fisher’s scientific) buffer tablets of pH 4.0, 7.0, and 9.2. As specified in the label of the buffer tablets, each buffer tablet was dissolved in distilled water to the volume mark of 100 ml volumetric flask. After the device has been calibrated, each honey sample was checked for its pH readings. The pH range of unadulterated honey should lie between the range of 3.5 to 5.5 [8]. Rheological Properties To make uniform treatment on viscosity, overall samples were maintained at 41±1℃ by using an electric air-heater. Viscosity was analyzed by viscometer (Model: DV-III Ultra Programmable Rheometer, Instrument Manufacturer: Brookfield) attaching spindle size of 63, the rheological properties of Honey were studied [1]. Optical Rotation For this experiment, 1.0% (w/v) of the Honey sample in distilled water was prepared at first. To the prepared sample, using blank correction as distilled water; the prepared sample was filled in the tube of polarimeter (Model: BK-P2S, Instrument Manufacturer: Biobase Biodustry [Shandong] Co. Ltd.) and its triplicate reading was taken. Honeydew honey should exhibit positive optical rotation while nectar honey exhibit negative optical rotation to the incident plane-polarized light [6]. By using the following equation, specific optical rotation was computed:- [𝛼]𝜆 𝑇 = 𝛼𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑏 × 𝑐 Where, [𝛼]𝜆 𝑇 = Specific Rotation in degree at T℃ and Light Wave-length (𝜆) 𝛼𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 = Observed Rotation in degree b = Path-length in the decimeter c = Concentration in gm/mL Relative Density Using the pycnometer (Glassware Manufacturer: Jain Scientific Glass Works [JSGW]), Digital Analytical Balance (Instrument Manufacturer: Bel Engineering), and laboratory thermometer, the density of our four (4) honey samples were determined. The room temperature of the lab during the time of the Experiment was also measured with the help of a Laboratory Thermometer. After that, an empty pycnometer was taken and its mass was noted using digital analytical balance. Now, it was filled with the honey sample and again its mass was weighed. The density of uncontaminated Honey typically ranges between 1.38 and 1.45 gm/ml [9]. 𝐷𝑒𝑛𝑠𝑖𝑡𝑦(𝜌) = 𝑊𝑡𝐹𝑖𝑙𝑙𝑒𝑑 − 𝑊𝑡𝐸𝑚𝑝𝑡𝑦 25 Where, WtFilled =the mass of Pycnometer after sample filling WtEmpty =the mass of the empty Pycnometer before filling Refractive Index The refractive index of the honey samples was measured by using Abbe Refractometer (Model: SN 4040, Instrument Manufacturer: Guru Nanak Instruments, New Delhi). Sample holding prism of the instrument was cleaned well by rinsing it, with the help of ethanol and soft tissue paper. After that, a drop of honey sample was then loaded into the lower Sample prism. Now, the upper sample prism was interlocked with the lower sample prism which facilitated contact between the two Sample prisms and ultimately formed a film layer of honey sample. By viewing through the eye-piece, coarse scale adjustment and fine scale adjustment knobs were rotated and readings were noted. Refractive index of unspoiled honey typically ranges from 1.474 to 1.504 indicating the presence of water content in honey from 25% to 13% respectively [10]. Pollen Contents Honey sample was prepared in a conical flask and it was left to shake in incubator shaker (Manufacturer: Biobase Biodustry [Shandong] Co. Ltd.) at 100 Revolution per Minute (RPM) for 15 Minutes at 45℃. The stock solution was then poured into the centrifugation tube. After that, it was allowed to centrifuge at 5000 RPM for 10 minutes in Centrifugation Apparatus (Model: NF 200, Manufacturer: Nüve Laboratories). The settled precipitate was scraped out and finally, it was observed in the microscope for determination of its shape and size. Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):3 2 - 3 9 Bajgain et al. 35 ©NJB, BSN 35 Qualitative Screening Test of Phyto- chemical Classes Plant metabolites were screened according to previously established methods [11-13] Total Phenolic Content Total phenolic content was determined by Folin- Ciocalteu (Thermo Fisher’s Scientific India Pvt. Ltd.) method, according to previously published method [12].The honey stock solution was prepared at 10% in water. A portion of 1 ml of the honey and 0.8 ml of 10 % aqueous reagent and followed by 2 ml of 15% sodium carbonate was added. Final volume was made by adding water. Mixture was incubated for 2 hrs, and absorbance was measured at 765 nm against the blank. A standard curve of gallic acid was prepare for quantification, using a concentration range of 250, 500, 750, 1000 and 1250 ppm and result were expressed as mg Gallic acid/Kg honey. Calculation of TPC was done using the following formula expressed in mg Gallic Acid Equivalent (GAE) per Kg of Honey sample, 𝑇𝑃𝐶 = 𝐺𝐴𝐸(𝑚𝑔/𝑙) × 𝑉(𝑚𝑙) × 10−3 (𝑙/𝑚𝑙) × 𝐷𝑓 𝑊𝑡𝑆𝑎𝑚𝑝𝑙𝑒 (𝑔𝑚) × 10 −3(𝑘𝑔/𝑔𝑚) where TPC =Total Phenolic Content (in mg GAE/Kg Honey) GAE = Gallic Acid Equivalent (in mg/l) V = Total Volume of Methanol Extract (in ml) Df = Dilution Factor Total Flavonoid Content Total Flavonoids content in honey was determined by a calorimetric method according to previous method. Briefly, 1.0 gm of honey sample was taken, which was dissolved in 10 ml 80% Ethanol to make sample stock solution. The sample stock solution was then allowed to incubate in incubator shaker (Biobase Biodustry [Shandong] Co. Ltd.) at 100 RPM for 15 Minutes at 45℃. Total 1.0 ml of supernatant sample stock solution was pipette out to which 0.2 ml of 10% (w/v) aqueous Aluminum Chloride was then added and subsequently, 0.2 ml of 1 M. Aqueous Potassium Acetate and 3 ml of 80% Ethanol was added. Finally, volume make-up was done to the mark adding sufficient Distilled Water and absorbance reading was measured at the wavelength of 415 nm against Blank Solution from the standard stock solution.[12].TFC was done using the following formula expressed in mg Quercetin Equivalent per Kg of honey; 𝑇𝐹𝐶 = 𝑄𝐸(𝑚𝑔/𝑙) × 𝑉(𝑚𝑙) × 10−3 (𝑙/𝑚𝑙) × 𝐷𝑓 𝑊𝑡𝑆𝑎𝑚𝑝𝑙𝑒 (𝑔𝑚) × 10 −3(𝑘𝑔/𝑔𝑚) where, TFC is Total Phenolic Content (in mg Quercetin/Kg Honey) QE is Quercetin Equivalent (in mg/l) V is Total Volume of Ethanol Extract (in ml) Df is Dilution Factor Anti-Oxidant DPPH Assay DPPH assay was estimated using the 2,2-diphenyl-1- picrylhydrazyl hydrate radical (DPPH) (Glentham Life Science Ltd, UK)) according to previous method of Kačániová[14 - 15]. The honey samples were diluted in Methanol at concentrations of 400, 800, 1200, 1600 and 2000 ppm solutions and from each dilution 0.3 ml was mixed with DPPH. The mixtures were vortexed, left in dark room temperature for 60 min and the absorbance was measured at 517 nm under UV-Visible spectrophotometer (UV 1800, Manufacturer: Shimadzu Scientific Instruments) correcting baseline Blank correction by Methanol. The Inhibition % is given by the relation 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100% Where AControl = mean Absorbance reading of 0 ppm solution against Methanol as Blank ASample =e mean Absorbance reading of 400, 800, 1200, 1600, and 2000 ppm solutions Results and Discussions Preliminary Purity Test Sand Sinking Test S1 took shortest period of time among all the samples while S4 took longer period of time to sink through Sand-hills. Various time (in Seconds) taken by samples to sink through the Sand surface is as shown in Table 2. Table 2. Time of Sand Sinking Test S.N. Sample Time (in Seconds) 01. S1 62.33±2.08 02. S2 197.33±2.52 03. S3 262.33±2.517 04. S4 900±0 Water Sinking Test All of our honey samples went down to the bottom of the cup without mixing up with the water except when stirred, which showed that all our honey samples complied the Water Sinking Test as mentioned in literatures mentioned above. Air Flow Test All the honey samples went down like a thread without breaking ranging 0.5 to 3 seconds as a continuous thread, which also showed that all our honey samples Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):3 2 - 3 9 Bajgain et al. 36 ©NJB, BSN 36 complied for the Air Flow Test as mentioned in literatures mentioned above. Confirmatory Purity Test Colour From colour shade analysis of Honey Samples with that of reference shade of Yellow colour, our different honey samples demonstrated varieties of colour as listed in Table 3. Table 3. Different Shades of honey as per base Yellow colour S.N. Sample Shade of Yellow Colour 1. S1 Amber 2. S2 Light Amber 3. S3 White 4. S4 Extra-Light White pH Presence of carbohydrate in dominant amount is the reason to which Honey shows slightly acidic nature. Due to the reason, Honey has a good Anti-Microbial potency. Among four samples, S3 showed highly acidic with pH of (4.467±0.0306) where S1 showed lowest pH with the value of (5.05 ± 0.02). Various values of pH shown by different honey samples are tabulated down in Table 4. Table 4. Various pH readings of different Honey Samples S.N. Sample pH 1. S1 5.05±0.02 2. S2 4.833±0.0379 3. S3 4.467±0.0306 4. S4 4.767±0.0416 Rheological Properties Among the samples, only S1 showed Newtonian type of flow property, while rest of the samples showed Pseudo-plastic flow property upon checking Rheological properties of Honey Samples using Brookfield Viscometer. Plot of Rotation per Minute (RPM) Versus Torque% and RPM Versus Viscosity (in centi-Poise) of different Honey Samples as shown in figure 3-6. Figure 3. RPM Versus Torque% and RPM Versus Viscosity (cP) (i.e. Rheological Properties of S1) Figure 4. RPM Versus Torque% and RPM Versus Viscosity (cP) (i.e. Rheological Properties of S2) 320 345 370 395 0 100 200 300 V is co si ty ( cP ) RPM S2 0 20 40 60 80 100 0 50 100 150 T o rq u e % RPM S3 0 20 40 60 80 100 0 50 100 150 T o rq u e % RPM S1 0 20 40 60 80 100 0 100 200 300 T o rq u e % RPM S2 0 50 100 150 200 250 300 0 50 100 150 V is co si ty ( cP ) RPM S1 Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):3 2 - 3 9 Bajgain et al. 37 ©NJB, BSN 37 Figure 5. RPM Versus Torque% and RPM Versus Viscosity (cP) (i.e. Rheological Properties of S3) Figure 6. RPM Versus Torque% and RPM Versus Viscosity (cP) (i.e. Rheological Properties of S4) Specific Optical Rotation From the literatures, it is established fact that Honeydew Honey and Adulterated Honey only shows positive Specific Optical Rotation, while Nectar Honey shows negative Specific Optical Rotation to the plane polarized light. S2 showed highest angle of rotation among the four samples while S1 showed positive angle of rotation. Various values of degree of Rotation are shown in Table 5. Specific Gravity Honey is denser than water with 1.3 to 1.4 folds. S3 and S1 are the respectively highest and lowest dense Honey Samples among the samples involved in this study. Table 6. shows different values of Specific Gravity which was obtained during analysis. Table 5. Specific Optical Rotations of different Samples at Concentration of 1.0% (w/v) in Water. S.N. Sample Specific Optical Rotation 1. S1 (+)5.75±0.4684 2. S2 (-)12.71±0.234 3. S3 (-)8.79±0.3098 4. S4 (-)2.299±0.3098 Table 6. Specific Gravity of samples observed at Room Temperature of 14℃. S.N. Sample Specific Gravity 1. S1 1.35±0.00017 2. S2 1.404±0.0016 3. S3 1.409±0.00022 4. S4 1.376±0.00497 Refractive Index and Moisture Content Moisture Content in Honey Samples is indicated by the Refractive Index values of Honey Samples. S1 has highest Refractive Index and thus Moisture Content and S4 has lowest Refractive Index and Moisture Content upon analysis of four different samples. Table 7 is listed with different observations of Refractive Index and Moisture Content of all four honey samples involved in the study. Table 7. Refractive Index and Moisture Content (in %) of Samples S.N. Sample Refractive Index Moisture Content (in %) 01. S1 1.473 25 02. S2 1.476 24.2 03. S3 1.476 24.2 04. S4 1.489 19 Figure 7. Microscopic View of Pollen Content at 400x Magnification of S1(1), ,S2(2), S3(3) andS4(4). Microscopic Studies of Pollen Contents Upon viewing microscopic slides of different honey samples, we found out the spring season harvested honey containing the same kind of pollen and it was different from autumn harvested sample. For the case of S4, as it is nectar honey of Diploknema butyraceae; the honey sample exhibit different kind of pollen contents in it, which is totally different from all of the honey samples. Figure 7 contains the pictures of Microscopic Slides of different Honey Samples. 100 150 200 250 300 0 50 100 150 V is co si ty ( cP ) RPM S3 0 20 40 60 80 0 50 100 150 T o rq u e % RPM S4 640 660 680 700 720 740 0 50 100 150 V is co si ty ( cP ) RPM S4 Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):3 2 - 3 9 Bajgain et al. 38 ©NJB, BSN 38 Secondary Metabolites Upon performing qualitative screening of secondary metabolites in honey samples; there was positive test for flavanoids, glycosides, saponins, tannins, reducing sugar, terpenoids, amino acids and proteins. There was negative test for alkaloids and phlobatanins. Table 8 shows the summary of results for all four honey samples as obtained from qualitative screening of secondary metabolites. Table 8. Qualitative Screening of Secondary Metabolites in different Honey Samples S.N. Name of Test S1 S2 S3 S4 01. Mayer’s Test for Alkaloid - - - - 02. Flavanoid Test +++ +++ +++ +++ 03. Browntoger’s Test for Glycosides + + + + 04. Foam Test of Saponins ++ ++ ++ ++ 05. Ferric Chloride Test for Tannins + + + + 06. Benedict’s Test for Reducing Sugar ++ +++ ++ +++ 07. Xanthoproteic Test for Amino Acids and Protein +++ +++ +++ +++ 08. Terpenoids Test +++ +++ +++ +++ 09. Phlobatannins Test - - - - - Negative, + Weak Positive, ++ Moderate Positive, +++ Strong Positive Quantitative Determination of Secondary Metabolites TPC and TFC Upon plotting of standard calibration plot of Gallic acid for TPC and Quercetin for TFC, we obtained following graphs as shown in figures with R2 of 0.9658 and 0.981 respectively for the linear regression keeping Concentration (in ppm) along X-axis and mean absorbance along Y-axis. Mean absorbance is the average of absorbance reading taken from triplicate data. Figure 8. Standard Calibration Curve of Gallic Acid for TPC Figure 9. Standard Calibration Curve of Quercetin for TFC Using above Standard Calibration Curve and linear Regression equation of line, TPC and TFC was found to be highest in S1 and lowest in S3. Table 9 shows the results of estimated TPC and TFC of different Honey Samples. Table 9. Quantitative Determination of Secondary Metabolites S.N. Sample TPC (gm GAE/ Kg Honey) TFC (gm QE/ Kg Honey) 01. S1 6.862884943 0.3531322456 02. S2 1.626269056 0.1607248345 03. S3 1.042708154 0.01675598334 04. S4 2.615476423 0.1883074849 TPC = Total Phenolic Content and TFC = Total Flavanoid Content Figure 10. Concentration Versus Inhibition% Graph from DPPH Assay (Anti-Oxidant of different samples at varying Concentration) Table 10. Results of Anti-Oxidant Assays (IC50 Values and Times Potency with the respect to Standard) S.N. Samples IC50 (in ppm) Times Potency 01. Quercetin 346.9112586 1.000 02. S1 649.6465517 0.534 03. S2 2353.956344 0.1475 04. S3 9867.161765 0.0353 05. S4 1390.792215 0.2497 y = 0.00338x + 0.09117 R² = 0.96580 0 0.2 0.4 0.6 0.8 1 1.2 0 100 200 300 M e a n A b so rb a n ce Concentration (ppm) TPC y = 0.0426x + 0.0519 R² = 0.9811 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 A b so rb a n ce Concentration (ppm) TFC y = 0.00348x + 47.73923 R² = 0.93397 y = 0.00733x + 32.74550 R² = 0.96365 y = 0.00272x + 23.16132 R² = 0.91245 y = 0.01824x + 24.63195 R² = 0.93571 y = 0.02076x + 42.78935 R² = 0.97686 20 30 40 50 60 70 80 90 300 1300 In h ib it io n % Inhibition Concentration [ppm] DPPH Assay S1 S2 S3 S4 Quercitin Extract from Allium cepa Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):3 2 - 3 9 Bajgain et al. 39 ©NJB, BSN 39 Anti-Oxidant DPPH Assay Upon plotting, Concentration (in ppm) Versus % Inhibition of different Honey Samples and similarly, Standard of Quercetin Extract extracted from Allium cepa, we obtained various IC50value from the plot, through which we became able to compare between the times potency value of Anti-Oxidant ability of different samples to that with Standard Quercetin Extract. From the observation, we found out that S1 had maximum potency with 0.534 times and S3 has minimum potency with 0.0353 times of standard Quercetin Extract from Allium Cepa. Figure 10 is the plot of inhibition concentration (in ppm) versus % inhibition of different Samples and Standards involved in the study. Table 10 is the result of IC50 value and times potency of different honey samples obtained from the calculation. Conclusions Different tests performed during this research complied with the various standard research articles published in different journals. Potency of honey is affected by the types of honey and seasons at which it has been harvested. Honey with lower anti-oxidant potency can be used in formulation of topical cosmetological products or as daily dietary supplement. Honey with higher values of anti-oxidant potency can be used in combination with other different active ingredients for the formulation of different therapeutic products. 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