BIBECHANA Vol. 22, No. 1, April 2025, 52-62 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Physicochemical, antioxidant, and antimicrobial study in Nepalese honey Amrita Adhikari, Nabin Kumar Thapa, Anita Gurung, Niranjan Parajuli∗ 1Centarl Department of Chemistry, Tribhuvan University, Kathmandu, Nepal ∗Corresponding author. Email: niranjan.parajuli@cdc.tu.edu.np Abstract Five honey samples, mostly from western Nepal, were analyzed for physicochemical proper- ties, TPC, TFC, antioxidant, and antimicrobial assays. The results were found to be within the acceptable range, while some samples had higher moisture, HMF, and total ash levels. The TPC was in the range of 8.420 to 9.920 mg GAE/g and the TFC was between 0.039 to 0.103 mg QE/g. The IC50 values were calculated of which Forest honey was found to have the lowest IC50 value i.e. 7.735 ± 0.008 mg/mL. Additionally, samples also showed a large zone of inhibition against four pathogens. Besides, the correlation between physicochemical param- eters, TFC, TPC, and antioxidant activity was established by using Pearson’s correlation and PCA. It displayed that the variables IC50, reducing sugar, HMF, and TFC in samples were positively correlated with one another and inversely with total ash (%), acidity as formic acid (%), and moisture (%). Keywords Physicochemical parameters, Antioxidant, Antibacterial. Article information Manuscript received: June 4, 2024; Accepted: January 16, 2025 DOI https://doi.org/10.3126/bibechana.v22i1.74118 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Bees naturally generate honey as a food item through the collection of either flower nectar from the family: Apidae [1] or the waste of insects that consume living plant parts and insect secretion. The honey sourced from living plant parts and in- sect secretion is commonly categorized as honey- dew honey [2]. Since ancient times, honey has been utilized to cure illnesses such as eye diseases, in- fections, asthma, and fatigue [3]. It is composed of minor amounts of organic acids, 15-17 % wa- ter, and approximately 80-85 % sugars, vitamins, proteins, phenols, and flavonols [4]. Mainly, fruc- tose, glucose, and sucrose are three kinds of car- bohydrates found in honey. In addition, honey contains nearly all minerals in minute amounts in- cluding potassium, sodium, phosphorus, sulfur, cal- cium, iron, manganese, magnesium, silicon, and copper. The most prevalent mineral element is potassium, which accounts for almost one-third of the overall amount [5]. The presence of potassium, cadmium, and nickel indicates honeydew honey [6]. It is suggested that these minor elements of honey are associated with various biological activities to exemplify antioxidant, antibacterial, antiviral, anti- inflammatory, antiulcer, antihypertensive, vasodila- tor, antihypercholesterolemic, and antitumor ac- tivity [7]. It contains various enzymes which in- clude diastase, invertase, catalase, glucosylcerami- 52 http://nepjol.info/index.php/BIBECHANA niranjan.parajuli@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v22i1.74118 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 53 dase, glucose oxidase, α-amylase, β-glucosidase, α- glucosidase, and proteases. Diastase breaks down starch into dextrin and maltose contributing to the honey quality, while invertase transforms su- crose into glucose and fructose [8]. Glucose oxi- dase is involved in the conversion of glucose to δ- gluconolactone, followed by hydrolysis of gluconic acid and hydrogen peroxide, providing honey with antimicrobial properties [9]. Honey could be the best choice for use in medic- inal value, as in recent times, the multi-drug resis- tance problem has arisen and this problem can be solved by the natural products singly or in combina- tion with drugs. Besides therapeutic benefits, some honey e.g. Mad honey can also pose toxicity leading to a serious food safety concern. Such toxicity and hallucinating effects are due to the honey bees col- lecting poisonous plants nectar like Rhododendron species, Coriaria arborea, and Tripterygium wil- fordii Hook F.. Mad honey contains natural plant poisons, such as pyrrolizidine, tutin, triptolide and grayanotoxins [10]. The chemical makeup, taste, color, and biological activities are primarily deter- mined by its floral origin, species of bees, climate, and geographic area, however can additionally be altered by the weather circumstances, as well as their handling, packing, and storage [11]. In Nepal, five honeybee species are found. Apis cerana and Apis mellifera are suitable for hive management and honey production whereas Apis laboriosa, Apis dor- sata, and Apis florea are wild species [12]. Among them, only Apis mellifera is an exotic species, while the remaining four species are indigenous to Nepal [13]. The majority of the honey produced in Nepal is the multi-floral origin, but some are also unifloral origins like chiuri, mustard, buckwheat, rudilo, sun- flower, and litchi honey, as well as honeydew honey from pine, spruce (Salle Maha), and oak (Dalle Maha) trees mostly in mountain region [12]. For the import and export of honey, the global standard is contained in the Codex Alimentar- ius. The Codex Alimentarius standard, European Honey Directive, and National Standard are the standards that set rules for food safety, regula- tion, and honey authenticity [14]. It has been re- ported that the various physicochemical parameters including moisture, sucrose, reducing sugar, total ash, acidity as formic acid, hydroxymethylfurfural (HMF), fructose/glucose ratio, etc are widely used to analyze botanical authentication of honey. In honey, glucose and fructose are present in higher amounts whereas twenty-two different compounds such as isomaltose, maltose, sucrose, etc are present in small amounts. Further, a nondigestible molecule (fructooligosaccharides) supports the growth of in- testinal bacteria and helps to enhance the human digestive system. Thus, it is also named as prebi- otics [15, 16]. Honey also contains low-fat, water- soluble vitamins and vitamin C in higher amounts, whereas the fat-soluble vitamins and vitamin B are present in small amounts [17]. Nepal has a vast array of honey types and huge potential for honey production, yet it has not fully reached its capabilities in honey harvesting due to limited research and investment in Nepal. Thus, a key goal of this study is to explore the physic- ochemical parameters, antioxidant properties, and antimicrobial test, and conduct principal compo- nent analysis on varieties collected which include local, market-sourced, and forest honey. 2 Materials and Methods 2.1 Chemicals We acquired quercetin (CAS No: 117-39-5) from Sigma-Aldrich in Germany. Gallic acid (CAS No: 5995-86-8) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (CAS No: 1898-66-4) were purchased from Molychem in India and HiMedia (India), respec- tively. Growth medium (MHA and MHB) were ob- tained from Sigma-Aldrich. Other chemicals were bought from Fisher Scientific (India). 2.2 Collection of honey samples Samples of honey were collected from multiple places, including Pokhara, Rukum, Lamjung, and Kathmandu (Table 1). The honey types stud- ied were Forest honey, Mad honey, Mustard honey, Chestnut honey, and Indian brand-marketed honey. 2.3 Physicochemical parameters The physicochemical parameters included by the International Honey Commission for the quality control of honey such as moisture, sucrose, reduc- ing sugar, total ash, acidity as formic acid, Hydrox- ymethyl Furfural (HMF), and fructose/glucose ra- tio were analyzed [18]. Moisture In honey, the moisture content was analyzed by op- erating the refractometric method with the help of an Abbe refractometer [19]. Total ash content Five grams of honey were subjected to a muffle fur- nace at 55 °C for five hours. Afterward, the ash was obtained by cooling it to room temperature and then weighed [20]. Acidity as formic acid Acidity as formic acid is calculated by using the titrimetric method [20]. Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 54 Hydroxymethylfurfural The UV-spectrophotometric method was employed to analyze and determine the levels of HMF [18]. Firstly, 25 mL water and 5 g honey were added to a 50 mL volumetric flask. Further, solution I and II of 0.5 mL carrez solution were added on, mixed, and diluted with water. After filtration, 5 ml of filtrate was taken in two test tubes. In one test tube, dis- tilled water of 5 ml was added, while the other test tube received 5 ml of a 0.2% sodium bisulfite solu- tion as the reference. The contents within each tube were effectively blended in a vortex mixer. Subse- quently, the sample’s absorbance was determined at 284 nm, and the absorbance of the reference was assessed at 336 nm. Finally, the HMF (mg/100 g) content was computed based on the measurements. Table 1: List of honey samples collected for the study from different regions of Nepal. S. N. Name of Sam- ple Floral Source Botanical Classification Floral Loca- tion Local Name 1 Mustard honey Mustard Brassica napus Pokhara Tori 2 Forest honey Forest Unspecified Rukum Vire Rukum 3 Mad honey Multi-floral Rhododendron arboreum and forest Lamjung Vire Pokhara 4 Indian brand marketed honey Market Unspecified Kathmandu Market honey 5 Chestnut Chestnut Castanea sativa Pokhara Katus pH The pH of honey was measured by a pH meter. 3 g of sample of honey was dissolved in 30 mL of water and mixed by agitating for 30 minutes. At ambient temperature, the pH of the samples was determined [21]. Reducing sugar The AOAC (2005) method was used to analyze re- ducing sugar content in honey [20]. 1 gm of honey was added in 10 mL lead acetate solution (20%) taken in a volumetric flask of 250 mL which was diluted up to the mark and filtered. The filtrate (25 ml) was combined with 100 mL of distilled wa- ter in a 500 ml volumetric flask. A few drops of 10 % potassium oxalate solution were added until no more precipitate formed. In a 250 ml conical flask, 5 ml each of Fehling A and B were combined with 10 ml of distilled water, boiled, and using the sample solution to titrate and find the amount of reducing sugar content. Sucrose The Fehling solution method was employed to de- termine the sucrose content in various types of honey [22]. The quantity of sucrose present was determined using the provided formula below: Sucrose % = (Total reducing sugars%−Reducing sugars%)×0.95 2.3.1 Glucose ratio The glucose percentage was determined using an iodometric method, and this amount was deducted from the percentage of reducing sugars to calculate the fructose percentage and the fructose-to-glucose ratio. The calculations were performed using the formulas provided below [23]. Glucose % = N of thiosulphate × (blank − titre) × a× 100 0.1 N × b 2× 0.1N × b where a=0.009005, b= weight pf sample Fructose % = Reducing sugar %−Glucose % Fructose : Glucose ratio = Fructose % Glucose % Impurities 50 mL of water was used to dilute each honey sam- ple of 10 g and blended in a shaker for about 30 min- utes. Filtration was done and the sample obtained was kept in the oven at 103 ºC for 10 minutes and the dried filter paper was weighed [21]. By using the formula the percentage of impurity was measured. I = m2 m1 × 100% ‘I’ represents the quantity of impurities (%), ‘m1’ represents the mass of the sample taken for analy- sis (g), and ‘m2’ represents the mass of residue left on the filter paper after drying (g). Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 55 2.4 Total Phenolic Content As per the study, the total phenolic content was determined using the Folin-Ciocalteu method with fewer adjustments [24]. Gallic acid was taken as a standard (1 mg gallic acid dissolved in 1 mL ethanol to create a gallic acid solution). It was serially di- luted ranging from 10 to 100 µg/mL to prepare so- lutions of different concentrations. For each honey sample, 5 mg/mL of honey solution was prepared by dissolving in 70 % ethanol, 20 µL (at a con- centration of 5 mg/mL) was mixed with 100 µL of Folin-Ciocalteu’s reagent (diluted 1:10 with water), and 80 µL of 1 M aqueous sodium carbonate solu- tion (Na2CO3 ). After about 30 minutes at ambient temperature, the mixture’s absorbance at 765nm was analyzed using a microplate reader. The ex- tracts’ total polyphenolic content was expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g) of the extract. This analysis was done in triplicate. 2.5 Total Flavonoid Content As outlined by Pham et al., the aluminum chloride colorimetric method was utilized to determine the total flavonoid content (TFC) with minor alterna- tions [25]. In brief, a concentration of 5 mg/mL of honey was made ready adding 70 % ethanol. Sub- sequently, 20 µL of each extract was combined with 100 µL of distilled water and 60 µL of ethanol. Sub- sequently, 10 µL of 10% AlCl3 and 10 µL of 1M CH3COOK were added to each well. Quercetin was used as the standard reference. After that, the mix- ture was left at room temperature for 30 minutes in the shade, after which the absorbance was mea- sured at 415 nm using a microplate reader. The overall flavonoid concentration within the samples was expressed as milligrams of quercetin equivalent per gram of dry weight (mg QE/g) of the extract. This analytical process was conducted in triplicate. 2.6 Antioxidant Activity The assessment of antioxidant activity was con- ducted by employing the stable radical 2,2-dipheny- 1-picrylhydrazyl (DPPH) [25]. 100 µL of DPPH so- lution in methanol (0.1 mM ) along with 100 µL of honey solution made in methanol at different con- centrations (1000, 500, 250, 125, and 62.5 µg/mL) were loaded into a 96-well plate. Then, it was kept in the shade for 30 minutes and the absorbance was recorded at 517 nm. Further, Quercetin was em- ployed as a standard. Here, a reduction from DPPH radical (purple) to diphenyl picryl hydrazine (yel- low) was observed at varying concentrations. The antioxidant used for the determination of the abil- ity to inhibit DPPH radical was obtained by the below equation: % scavenging = A0 −At A0 × 100% Here, “Ao” represents the absorbance of control, while “At” corresponds to the absorbance of DPPH when exposed to a test or reference sample. 2.7 Antibacterial Activity The antibacterial activity of honey samples at var- ious concentrations (10% W/V, 20% W/V, 50% W/V, and 75% W/V) against four distinct bacte- rial strains ( namely: E. coli, S. aureus, S. shigella, K. pneumoniae) was examined through the agar well diffusion method on MHA plates [26]. A 0.5 McFarland equivalent of microorganism inocu- lum in Mueller Hinton broth (MHB) was spread on the MHA plate’s surface using an autoclaved cotton swab. Wells were bored by the clean and well-sterilized cork-borer in cultured MHA media. Then, a concentration of 100mg/mL of honey ex- tracts was filled into the wells. For the positive and negative control, neomycin and distilled water were used respectively and incubated at 37ºC for 18 to 24 hours. Finally, the zone of inhibition (ZOI) was observed and measured by a ruler in mm. 2.8 Statistical Analysis The MS-Excel was used to do a Karl Pearson corre- lation between the variables such as physicochem- ical parameters, TPC, TFC, and antioxidant ac- tivity. Additionally, to correlate several physico- chemical data (such as moisture content, sucrose, reducing sugar, total ash, acidity as formic acid, HMF, fructose/glucose ratio, and pH), TPC, TFC, and antioxidants. Further, using R studio Principal Component Analysis (PCA) was performed. PCA is a dimensionality reduction technique that trans- forms a set of numerous variables into a smaller set that retains most of the information from the original large data [27]. Pearson correlation helps to determine the degree and direction of correla- tion. Table 2 shows the degree of correlation as follows [28]. Table 2: The degree of correlation is represented by the numerical values. Amount of r Strength of the Correlation 0.0 < 0.1 No correlation 0.1 < 0.3 Low correlation 0.3 < 0.5 Medium correlation 0.5 < 0.7 High correlation 0.7 < 1 Very high correlation Note: ‘r’ indicates Karl Pearson’s correlation coefficient. Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 56 3 Results and Discussion 3.1 Physicochemical-parameters-1 The following Physicochemical parameters of all samples were determined and the results were men- tioned below (Table 3). 3.1.1 Moisture The standard set by the Codex Alimentarius must be below 20%. The honey sample above the set standard will undergo deterioration by fermenta- tion [29]. The water content of mustard, market, and chestnut honey fell within the accepted range, while forest honey (24.5%) and mad honey (25.7%) exceeded the standard range. The level of moisture is determined by factors like the relative humidity of the plant source, processing techniques, and storage conditions [30]. Moisture plays a crucial role in reg- ulating microbial growth and the potential spoilage of honey through fermentation [31]. 3.1.2 Total ash content The total ash content in samples is useful for the identification of honey. Here, the total ash content was from 0.1 to 0.7 in which all samples were within the standard limit except Forest honey with a 0.7 value and the lowest shown was market honey [32]. Out of five samples, one sample (forest honey) was out of range. 3.1.3 Acidity as formic acid Inorganic ions, organic acids (sulfate, chloride, and phosphate), esters, and lactones are present in honey as free acidity[30]. Vitamin C, protein, and phenolic acids are the proton donors and help in identifying the quality of honey. As the honey wors- ens its free acidity increases and fermentation is seen by the conversion of sugar to organic acids. Acidity as % formic acid consists of 0.001 to 0.01%. The lowest value of acidity as % formic acid is of Indian brand market honey and the highest is of Mustard and Forest honey which are present in an acceptable range. 3.1.4 Hydroxymethylfurfural (HMF) The HMF of various samples was from 2 to 87 mg/kg of which the lowest was that of Forest honey and the highest was of Indian brand market honey which exceeded the given standard value. HMF lev- els serve as a common measure for assessing the freshness of honey, as it is typically undetectable or found in minute amounts in newly harvested kinds of honey. However, its content tends to increase due to processing and aging [33]. Indian brand mar- ket honey is processed honey and is made available in the market after a prolonged period, this could potentially account for the elevated levels of HMF observed in market honey. 3.1.5 Reducing sugar The reducing sugar of different samples of honey was from 63.8 to 74.3 %, in which the highest amount of reducing sugar was present in market honey and the least in Mad honey. The reduced sugar content in all the analyzed samples is up to the standard range. The amount of sugar present in honey is influenced by factors such as the source of nectar from different types of flowers, the prevailing climate, additionally the methods used in handling and keeping [34]. 3.1.6 Sucrose Among the five honey samples, the sucrose content ranged from 0.1% to 7.2%. The lowest sucrose con- tent (0.1%) was found in Mad honey, while the high- est content (7.2%) was observed in chestnut honey. The sucrose content of different honey from differ- ent places was within the standard range ( 10). 3.1.7 Fructose/Glucose ratio The chestnut honey exhibited a fructose/glucose ra- tio of 1.34, while the other honey samples showed a ratio of 1.2. The carbohydrate composition of honey is responsible for its capacity to retain mois- ture, prolong shelf life, react in microwaves, and enhance the development of colors and flavors. The fructose-to-glucose ratio determines the ability to crystallize honey. Honey crystallizes slowly if the fructose/glucose ratio exceeds 1.3 whereas it occurs quicker when the proportion is less than 1.0 [35]. 3.1.8 pH The honey samples’ pH values varied from 3.26 to 5.80 depending on organic acids and these organic acids are responsible for the aroma and antimicro- bial activity of honey. All the honey samples were within the standard pH range except the chestnut honey. The pH difference is based on the maturity period, the concentration of minerals, and chemi- cal composition [36]. The pH of the floral and wild honey in this research work shows a resemblance with the range of pH in a study by Alves et al. [37]. 3.1.9 Impurities The impurities percentage in the samples was in the range of 1.11%-6.77%. The highest impurities were found in market honey. This might be due to the adulteration, additives, and inclusion of other substances during processing. Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 57 Table 3: Physicochemical parameters and impurities in five honey samples. S.N. Name of Sample Moisture (%) Total Ash (%) Acidity as Formic Acid pH Impurity (%) HMF Reducing Sugar (%) Sucrose (%) Fructose/Glucose Ratio 1 Mustard honey 21.3 0.2 0.01 3.26 2.50% 40 68.5 3.4 1.2 2 Forest honey 24.5 0.7 0.01 4.22 4.10% 2 63.9 0.2 1.2 3 Mad honey 25.7 0.4 0.004 4.42 4.66% 12 63.8 0.1 1.2 4 Market honey 19.2 0.1 0.001 4.78 6.77% 87 74.3 1.0 1.2 5 Chestnut honey 20.2 0.4 0.004 5.80 1.11% - 68.1 7.2 1.34 6 Standard Range ≤ 23 ≤ 0.5 - 3.5-5.5 - ≤ 40 ≥ 60 ≤ 10 ≥ 0.95 3.2 TPC, TFC, and Antioxidant Activity The TPC, TFC, and free radical scavenging ac- tivity were assessed in various honey extracts, re- vealing TPC value in a range of 8.420 ± 0.703 to 9.920 ± 0.649 mg GAE/g, while TFC value was less than 1 mg QE/g in all the analyzed honey samples. Among the collected samples, the antioxidant activ- ity (based on IC50 value) of honey samples followed the order: Forest honey > Mad honey > Chest- nut honey > Mustard honey > Indian brand Mar- ket honey. In comparison to the standard quercetin (IC50 value = 0.34 ± 4.11), all analyzed honey sam- ples displayed lower antioxidant activity. These val- ues are compared with data from similar studies on honey’s antioxidant properties and found similar re- sults [38–41]. The TPC, TFC, and antioxidant ac- tivity (IC50 values mg/mL) are displayed in Fig 1, Fig 2, Fig 3, and Table 4. Table 4: TPC, TFC, and IC50 values of different honey samples. Honey Samples TPC (mg GAE/g) TFC (mg QE/g) IC50 (mg/mL) Mustard honey 8.976± 1.395 0.053± 0.004 11.628± 0.004 Forest honey 9.809± 1.345 0.039± 0.011 7.735± 0.008 Mad honey 9.106± 1.473 0.044± 0.008 10.2± 0.002 Indian brand market honey 9.920± 0.649 0.103± 0.066 12.51± 0.003 Chestnut honey 8.420± 0.703 0.055± 0.012 10.92± 0.005 Quercetin - - 0.34± 4.11 Figure 1: Bar diagram of TPC of honey samples. Figure 2: Bar diagram of TFC of honey samples. Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 58 Figure 3: The plot of % inhibition of DPPH Vs con- centration of different honey samples. 3.3 Correlation of bioactive compound and antioxidant activity We observed the normal distribution of the response variable (antioxidant activity) (p>0.05) using the Shapiro-Wilk normality test [42] The linear corre- lation between the two variables was assessed by using the Karl Pearson correlation coefficient. The results of the correlation between the variables are provided in Table 5. The moisture (%) exhibited a moderately neg- ative relation with sucrose and HMF, while it dis- played a strongly negative relationship with reduc- ing sugar content (%), TFC, and IC50 value. The sucrose (%) had a minimal connection with acid- ity in the form of formic acid (%) and TFC. Su- crose (%) demonstrated a highly negative corre- lation with TPC (-0.816), but a notably positive correlation with the ratio of Fructose/Glucose ratio (0.897). The percentage of reduced sugar exhibited a positive correlation with TFC, HMF, and IC50 values. The proportion of total ash (%) showed no significant correlation with the fructose/glucose ra- tio, TPC, and pH. However, it revealed a strong negative correlation between TFC and IC50 value. HMF displayed a markedly positive correlation with TFC and IC50 values. HMF has an extremely strong positive relationship with the TFC (0.909). The relationship between TPC and TFC is pos- itive, suggesting that most of the phenolic com- pounds in these honey types are flavonoids. In the provided Table 5, TPC and TFC exhibit a mod- erately negative correlation with the IC50 value for DPPH free radical scavenging. This implies that higher TPC and TFC levels correspond to lower IC50 values, indicating that as the phenolic and flavonoid content increases, the honey’s ability to neutralize free radicals improves. This connection between phenolic/flavonoid content and antioxidant activity aligns with the findings of numerous stud- ies. Conversely, the correlation between TFC and IC50 value (a measure of the level of antioxidants) is positive according to the table. This suggests that antioxidant activity isn't solely attributed to flavonoids but might be influenced by other fac- tors such as HMF, enzymes, proteins, and mi- nor constituents. In certain instances, flavonoids might even interact with other compounds that pos- sess antioxidant properties, potentially diminishing overall antioxidant effectiveness. Similar outcomes have been observed in prior honey-related research [43–45]. Table 5: Karl Pearson’s correlation between physicochemical parameters, TPC, TFC, and IC50. Correlation Moisture (%) Sucrose (%) Reducing Sugar (%) Total Ash (%) Acidity as formic acid (%) HMF (mg/kg) Fructose/Glucose ratio TPC (mgGAE/g) TFC (mgQE/g) pH IC50 Moisture (%) 1 Sucrose (%) -0.577 1 Reducing Sugar (%) -0.907 0.209 1 Total Ash (%) 0.685 -0.160 -0.827 1 Acidity as formic acid (%) 0.390 -0.058 -0.548 0.502 1 HMF (mg/kg) -0.605 -0.253 0.873 -0.828 -0.447 1 Fructose/Glucose ratio -0.395 0.897 0.049 0.097 -0.250 -0.431 1 TPC (mgGAE/g) 0.097 -0.816 0.216 0.036 -0.027 0.520 -0.743 1 TFC (mgQE/g) -0.759 -0.027 0.952 -0.767 -0.696 0.909 -0.083 0.400 1 pH -0.315 0.489 0.174 0.093 -0.670 -0.195 0.792 -0.304 0.227 1 IC50 -0.752 0.326 0.833 -0.980 -0.573 0.742 0.099 -0.180 0.754 0.076 1 3.4 Principal Component Analysis of Dif- ferent Variables In this analysis using Principal Component Anal- ysis on the dataset, 50.4% of the variation is ex- plained by the first principal component (PC1), and 31.4% is explained by the second principal compo- nent (PC2). The scree plot, shown in Fig 4, visu- ally displays the percentage of variation represented by each principal component. It reveals that there are two prominent principal components that, when combined, account for 81.8% of the total variance present in the five distinct samples of honey. The two-dimensional graph of two prominent principal components that includes 81.8% of the total varia- tion of the data is shown in Fig 5. Fig 5 is a variable correlation plot that shows the relationship between the variables of honey samples. HMF, TFC, reducing sugar, and fruc- tose/glucose showed a better representation of vari- ables. Reducing sugar, TFC, and HMF again fruc- tose/glucose ratio, pH, and sucrose are positively correlated. Kowalski et al. also showed a positive correlation with fructose/glucose ratio, pH, and su- crose[46]. IC50 is highly positively correlated with each other and negatively correlated with Total Ash (%), acidity as formic acid, and Moisture (%). To- tal ash and f/g ratio, acidity as formic acid, and TPC are approximately right-angled indicating no correlation between them. https://quantifyinghealth.com/report-shapiro-wilk-test/ Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 59 A PCA biplot is shown in Fig 6, Honey samples Mustard, Chestnut, Forest, Mad, and market honey were given by points 1, 2, 3, 4, and 5 respectively and the variables were represented by the vectors. Based on this biplot, Points: 2, 4, and 5 have a ma- jor impact on the variability of the data and in prin- cipal components, and observations 2 and 3 share similarities in terms of their variability in their re- spective datasets. The components of biplot Dim 1 and 2 gave 81.8% of the variance in the descrip- tive profile of five different samples. Kowalski et al. showed the first two components of 68.67% of the variance [45]. Figure 4: Scree plot of the principal components. Figure 5: The principal component plot of vari- ables. Figure 6: Principal component biplot of observa- tions and variables. 3.5 Antibacterial Activity The agar well diffusion method was employed to as- sess the susceptibility of four bacterial strains (E. coli, S. aureus, S. shigella, and K. pneumoniae) to various concentrations of samples, as outlined in Table 6. The largest zone of inhibition (ZOI), mea- suring 36 mm, was observed at a 50% concentration of two honey samples (Chestnut honey and Forest honey) against E. coli. Mustard honey and Market honey exhibited no significant impact on E. coli. However, market honey demonstrated the highest inhibitory effect against the other three bacterial species: S. aureus (34 mm), S. shigella (35 mm), and K. pneumoniae (30 mm). Mustard honey was also ineffective against S. aureus but showed a mod- erate effect against S. shigella and K. pneumoniae. This study found antimicrobial activity at 50% v/v of sample concentration of almost all samples to the four microorganisms (E. coli, S. aureus, S. shigella, K. pneumoniae). Fj et al. have also reported simi- lar antimicrobial activity in all the analyzed honey samples at a concentration greater than 60% v/v against the four different microorganisms (E. coli, S. aureus, P. aeruginosa, and B. subtilis) [46,47]. In general, the zone of inhibition tended to be slightly larger for gram-negative bacteria in con- trast to gram-positive bacteria. This divergence in results can be attributed to the structural charac- teristics of the bacteria and their distinct mecha- nisms of toxicity. The major antibacterial element in honey is primarily hydrogen peroxide which dis- turbs the structure and function of bacteria result- ing in their diminished viability due to the oxida- tion of their constituent molecules [48]. The relative amounts of glucose oxidase, which bees create, and catalase from flower pollen affect its levels [9]. Amrita Adhikari et al./ BIBECHANA 22 (2025) 52-62 60 Table 6: Zone of inhibition of honey samples at different concentrations. Samples E. coli (mm) S. aureus (mm) S. shigella (mm) K. pneumoniae (mm) 10% 20% 50% P.C. 10% 20% 50% P.C. 10% 20% 50% P.C. 10% 20% 50% P.C. Chestnut honey 12 22 36 14 8 8 9 12 12 24 26 13 0 9 10 16 Forest honey 18 20 36 18 0 0 0 10 0 28 29 12 25 28 28 15 Mad honey 10 22 32 19 12 22 25 0 0 22 28 11 0 0 0 0 Mustard honey 0 0 0 0 0 0 0 23 22 28 30 22 25 25 30 20 Market honey 0 0 0 0 18 22 34 23 24 28 35 20 28 30 30 20 4 Conclusion A comprehensive examination of five honey sam- ples gathered from diverse regions of Nepal was con- ducted and considerable variability was found in the physicochemical parameters, TFC, TPC, antioxi- dant activity, and antibacterial activity. It might be due to unique geographical regions and botani- cal origins. The pH and impurities of all the sam- ples examined were within the range of the qual- ity standard except for moisture (due to premature harvest), ash content (indicates honeydew honey), and HMF (due to processing and storage condi- tions). Indian brand market honey displayed com- paratively lower antioxidant properties among the samples, possibly due to ingredient loss during pro- cessing and packaging, and exhibited lower antiox- idant activity. The samples were very efficient in opposition to the tested gram-negative bacteria dis- playing a high zone of inhibition, 36 mm at a 50% concentration of two types of honey (chestnut honey and Forest honey(R) against E. coli. Market honey exhibited noteworthy inhibitory effects against S. aureus (34 mm), S. shigella (35 mm), and K. Pneu- moniae (30 mm). The findings of this study demon- strated the important physiochemical parameters, TPC, TFC, and potential of honey ranging from its antioxidant properties to its antimicrobial ac- tivity, suggesting it is a natural therapeutic agent. Further study is necessary to identify the bioactive substances, their particular targets, and their mech- anisms of operation as antioxidant and antibacterial agents. Abbreviations MHA Muller Hinton Agar TPC Total Polyphenol Content TFC Total Flavonoids Content GAE Gallic Acid Equivalent QE Quercetin Equivalent DMSO Dimethyl Sulphoxide DPPH 2, 2 Diphenyl-1-picrylhydrazyl IC50 Concentration provides 50% Inhibition MHB Muller Hinton Broth ZoI Zone of Inhibition mg Milligram µL Microliters DFTQC Department of Food Technology and Quality Control IHC International Honey Commission MGO Methylglyoxal HMF 5-hydroxymethylfurfural PCA Principal Component Analysis F/G(f/g) Fructose/Glucose ratio CFU/mL Colony-forming unit per milliliter MDR Multidrug-resistant ROS Reactive oxygen species Conflicts of Interest The authors declare no conflicts of interest. Authors contributions Conceptualization, N.P.; methodology, A.A.; writing-original draft preparation, N.K.T, A.A.; su- pervision, N.P, Review, A.G.; All authors have read and agreed to the published version of the manuscript. 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Introduction Materials and Methods Chemicals Collection of honey samples Physicochemical parameters Glucose ratio Total Phenolic Content Total Flavonoid Content Antioxidant Activity Antibacterial Activity Statistical Analysis Results and Discussion Physicochemical-parameters-1 Moisture Total ash content Acidity as formic acid Hydroxymethylfurfural (HMF) Reducing sugar Sucrose Fructose/Glucose ratio pH Impurities TPC, TFC, and Antioxidant Activity Correlation of bioactive compound and antioxidant activity Principal Component Analysis of Different Variables Antibacterial Activity Conclusion