Introduction

The honey has been described as Madhu, Makshika by the Ayurveda scholars with different nutritional and medicinal values. Although its description is also found in different traditional systems of medicine in the world.^1-3 However, Ayurveda has described eight varieties of honey with different medicinal properties produced by eight types of honey bees. But very few scientific research studies have been done on the varieties and properties of honey as well as there are no studies on the eight varieties of honey as mentioned by the scholars of Ayurveda. Honey (Madhu) is one of the natural untreated food productsproduced from nectar by honey bees. It is sweet in primary taste and astringent in secondary taste; dry, cold, kindle digestion, good for color and voice, light (easily digestible), bestows softness to the body, is scarificant, good for heart and eyes, pro-constipating, enters into minute channels, reduces fat(Meda); indicated in diabetes, hiccup, dyspnoea, cough, diarrhea, vomiting, thirst, worms infestation and poison; alleviates all the three Doshas -mitigates Kapha by its easy digestibility Vata and Pitta by its sliminess, sweetness, and astringency. Honey aggravates Vata, is heavy, cold, alleviates disorders of Rakta, Pitta, and Kapha; is union-promoter, expectorant, rough, astringent, and sweet.^{4, 5} Honey, if heated or taken by a person suffering from heat becomes fatal, due to its association with poisons. It is useful in small quantities due to its properties- heavy, rough, astringent and coldproperties.^{4, 6} Honey has many nutrients and acts as an immunomodulator (LehanKarma),^7-9 gives the effect of the drug with which it combines(Yogavahi),^{10,  11} adjuvant to enhance drug absorption or bioavailability of drug (Anupana),¹² antibiotic,^13-15 anti-oxidant,¹⁶  anti-fungal,^17-21 anti-inflammatory,¹  use in the management of diabetes (Prameha),⁵  obesity (Sthaulaya)^{5, 12} and dyslipidemia,²²  etc.

Ayurveda has also mentioned certain factors such as time, the vessel of storage, combination with food, exposure to heat, with which there are changes in properties of honey and its effect on the body. Just after the procurement, honey is named as new honey(NavaMadhu) and after the passage of one year of time is called old honey (Purana Madhu). The properties and therapeutic uses of new and old honey are different (Table 1), so to get the desired effects the time of collection of honey and type of honey should be considered and advised accordingly.

Table 1: Effects of new (Nava) and one year old (Purana) honey on Body¹¹

Honey is produced all over the world, but the composition of honey and properties of honey is influenced by a number of factors such as geographical origin, botanical (flower) sources of nectar, environmental and climatic conditions as well as its harvesting, processing and storage techniques.^{23,  24} The various varieties of honey may be grouped by the floral source; monofloral  or multi-floral and bee species like Apis dorsata, Apis melifera, Apis crena, etc. The monofloral  honey means dominating pollen grain originated from only one particular plant and multi-floral honey means no dominant pollen type in the sample.²⁵

Kinds of Honey

In Ayurveda, eight varieties of honey with specific properties have been described by different Scholars of Ayurveda viz.Pauttika, Bhramara, Kshaudra, Makshika, Chatra, Aarghya, Auddalaka,and Dala.²⁶ But according to Charak Samhita, four types of honey i.e. Makshika, Bhramara, Kshaudra, Pauttika, derived from specific honey bee called Makshika, Bhramara, Kshaudra,Puttika respectively.^{5, 10}

Characteristics of Pauttika Honey

Honey prepared by Puttika bees is called as Pauttika honey, which resembles like ghee (in color and consistency). As per the commentary of Chakrapani have noted that “pidingala makshika mahetvah puttikah, tudhdvam pauttikam” Puttika bees are big in size and yellowish in color²⁷ which lives inside hollows of big trees. This type of honey is hot in potency, causes dryness, aggravation of Pitta, Rakta and Vata, burning sensation, heartburn during digestion, cures diabetes,⁵ etc.

Characteristics of Apis dorsata

Apis dorsata is a type of honey bee also called the giant honey bee, found mainly in forested areas of South and Southeast Asia. It is similar to Apis mellifera in appearance but is larger in size. They are about 17-20 mm in length, thus they are known as the “giant” honey bees. These show aggressive behavior against any disturbance.

The entomologist Michael S. Engel has identified the four subspecies of Apis dorsata found in different places of the world. The subspecies Apis dorsata dorsata (Indian giant honey bee) is primarily found in India. They build single large nests up to 150 cm in length and in open places on branches of high trees or buildings.

It is considered the most defensive of all of the honey bees with sharp sting up to 3 mm long and easily penetrate clothing and even the fur of a bear and are more defensive than the African honey bees.^{28, 29}

Various studies have reported significant differences in the physico-chemical properties, nutritional values, appearances, color and sensory parameters in new and old honey and suggest about the differences in effects after intake new or old honey.^30-36 In this manuscript, the attempt has been made to analyze and find out the differences in new and old honey properties in the Pauttika type of honey on standard scientific parameters.

Materials and Methods

1. Collection and storage of honey
2. Identification of honey

• Identification of bee specimens to ascertain the type of honey bee.
• Organoleptic evaluation as per descriptions in Ayurveda about the specific type of

3. To standardize the quality of honey

• Physico-chemical analysis
• Minerals analysis
• Sensory analysis using the hedonic scale
• Color (hunter color lab) of honey
• FTIR

Chemical and Reagents

All the chemicals and reagents used were of the analytical grade and as per the standard method for the analysis of honey. The analysis and reporting of the honey samples were  carried out at the Department of Dairy Science and Food Technology,Institute of Agricultural Sciences, BHU, India.

Source of Collection and Storage of Honey

The present study was carried out on honey procured from the region Uttar Pradesh, India. To ensure the authenticity of the honey sample, the collection was made directly from hives nested onthe Bamboo tree, Village Karim Patti, Tanda, Distt. Ambedkar Nagar in the month of March, 2018.

The procured part of the hives was collected from groves, by pressing and squeezing in the traditional method of the combs containing honey. It was filtered before storage or use at room temperature. Filtered honey was stored for one year in an airtight and light-resistant glass container for the study.

Identification of Honey and its Bee

Bee Specimen Identification

At the time of procurement of honey from natural honey hives nested on a tree, few honey bees have been also collected and preserved by standard method for identification of honey bee, so that the determination of the type of honey can be done as described in Ayurvedaand standard method.

The bees were killed using ethyl acetate after that specimens were dry mounted[³⁷]and the identification of Specimens of honey bees was done by the expert entomologist in the Division of Germplasm Collection and Characterization, ICAR-National Bureau of Agricultural Insect Resources (NBAIR), H.A. Farm Post, P.B. No 2491, Bellary Road, Bengaluru- 560024, Karnataka, India.

Organoleptic Evaluation

The organoleptic evaluation was done two times, first at the time of procurement of fresh honey sample and second after the passing of one year.

The characteristic of honey  described in Ayurveda was evaluated on organoleptic parameters such as color, odor, consistency, taste, etc. In Charak Samhita, four types of honey have been mentioned viz. Makshika, Bhramara, Kshaudra,Pauttika, which produced from specific honey bees calledMakshika, Bhramara, Kshaudra,Puttika respectively.^{5, 10} Amongst them, Pauttika honey resembles like ghee (in color and consistency). Puttika bees are big in size and yellowish in color.²⁷

Physico-Chemical Analysis

Ash content, acid insoluble ash, viscosity, moisture content, pH, acid value, and total soluble solids were determined as per the method described by the Association of Official Analytical Chemists (AOAC, 1990).³⁸ Reducing sugar content of the honey sample was determined as per the method recommended by Miller et al., 1959.³⁹ Hydroxyl methyl furfural (HMF) content in honey was determined by following the method of Keeney and Bassette, 1959 with slight modifications,⁴⁰ the Total Phenolic Content by the method involving Folin-Ciocalteau reagent and Gallic acid standards Hinneburg et al., 2006.⁴¹ Antioxidant activity- Determination of the antioxidant potential by the DPPH inhibition method as per the procedure is given by Mimica-Dukic et al., 2004, with slight modifications,⁴² the vitamin C content by Plummer TD, 1978 using 2,6-di- chlorophenolindophenol with minor modifications and was expressed as mg ascorbic acid/kg honey.⁴³

Minerals Analysis

Analysis of minerals and heavy metals was done as per AOAC, 1990using Flame Atomic Absorption Spectrophotometer (FAAS), Model AA-7000, Shimadzu, Japan except for P, which was analyzed spectrophotometrically as per AOAC, 1990.³⁸

Hedonic Rating Scale

The honey sample was analyzed for different sensory characteristics like color and appearance, body and texture, aroma and taste and overall acceptability. Sensory evaluation was performed by a panel of 9 semi-trained judges from the Department of Dairy Science and Food Technology, Institute of Agricultural Sciences, BHU, India. Sensory evaluation was done at 27 °C and 60% relative humidity. 9-point hedonic rating scale (1 = dislike extremely, 9 = like extremely), Amerine et al., 1965⁴⁴ was used for color and appearance, consistency, flavor, mouthfeel and overall acceptability.⁴⁵

Color Measurement

L-a-b values for honey using Hunter color Lab. ColorFlex EZ model was used calibrated first using a white tile and black tile. It measures color using Three‐dimensional scales, such as CIE L*a*b*, which has been developed to objectively quantify color values.  This scale defines color as follows:  L* (lightness) axis: black to white (0 to 100), a* (red-green) axis: positive values are red; negative values are green; 0 is neutral and b* (yellow-blue) axis: positive values are yellow; negative values are blue; 0 is neutral. All visible colors can be quantified within this 3‐D rectangular space.⁴⁵

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis

Infrared spectra of the honey sample was recorded using the PerkinElmer spectrum version 10.4.3 FTIR spectrophotometer. A pinch of honey has been taken and placed over the crystal present on stage. The IR spectrum was scanned between 4000 to 400 cm^-1 and transmittance was recorded. Before scanning the sample, the background signal was also recorded. The peaks thus obtained were matched against the IR interpretation chart and the functional groups were noted.

Results and Discussion

Identification of Honey Bee and Organoleptic Evaluation

On naked eye inspection, the color of the bees was yellowish, black. The entomologist has identified the specimen’s honey bees as Apisdorsata.

As per Ayurveda, the Puttika bees are big in size and yellowish in color,²⁷ which lives inside hollows of big trees. Thus, the characteristics found in the sample of honey bees, are similar to Apisdorsata as per the characteristics described in Ayurveda and the report of the entomologist. The physical characteristics of the honey samples were also the same as the characteristics of honey produced by Puttika honey bees as the color and consistency of honey was similar to ghee.

Thus, researchers  have determined that the collected honey was Puttika type of honey on the basis of similarity of characteristics of honey as well as sample honey bee (Figure 1, 2, 3, 4 & 5).

Figure 1: Honey bees specimen. Click here to View figure

Figure 2: Honey bee (Apis dorsata)  Click here to View figure

Figure 3: Fresh honey  Click here to View figure

Figure 4: Old honey  Click here to View figure

Figure 5: Size of honey bees. Click here to View figure

Picture of Honey Bee (Apisdorsata) and Honey (New and Old).

Tables (2, 3, 4 & 5) show the mean values and standard deviation of the basic characteristics of the physico-chemical analysis, AAS to obtain mineral contents, sensory evaluation and color appearance of honey. The obtained data could be discussed as the following:

Physico-Chemical Analysis of Honey

To standardized and validate the Pauttika honey, physico-chemical characteristics of the freshly tested Pauttika honey is compared with one-year-old Pauttika honey (Table 2) along with previous studies. Codex Alimentarius Commission (CAC, 2001) was used as a standard for comparison.

Table 2: Physico-chemical properties of fresh and old (one year stored at room temperature) Pauttika honey sample.

(TSS: total soluble solids, HMF: hydroxyl methyl furfuraldehyde, TPC: total phenolic content, N: number of replicates, Mean ± standard deviation values)

Ash content and Acid Insoluble Ash

The obtained ash values were 0.28% in fresh honey and 0.47% in old honey, which indicates the cleanness of the product. However, since these values were less than the limits allowed by CAC for floral honey (≤0.6%). Samat et al., 2017 and Islam et al., 2017 reported the ash content in fresh honey 0.14± 0.01%⁴⁶ and 0.14±0.0%⁴⁷ respectively. Another study on big honey has shown almost similar ash content i.e. 0.41±0.037%⁴⁸ as the one-year-old honey value in this study. The obtained acid insoluble ash values were 0.12% and 0.13% in fresh and old honey, respectively.

Viscosity

The value of viscosity was 102.9 (cP) (at 20 rpm) 37^oC in freshly procured Pauttika honey and 100 (cP) in old honey (Table 2), while other studies have reported 0.76 ± 0.22 (Pa·s) in Tualang honey and 0.53 ± 0.06 (Pa·s) Gelam honey.⁴⁹ Viscosity is one of the most significant physical and sensory characteristics of honey, that affects the quality and influenced by temperature, moisture content, as well as the presence of crystals and colloids in the product.⁵⁰

Moisture

The obtained moisture content was 25.58% in the fresh honey and 19.20% in the one-year-old honey (Table 2). As per the international regulations CAC (2001) the acceptable, good quality of the honey^{51, 52} contains the maximum value of moisture is ≤21%. But a study of S.P. Kek et al., 2018 have shown higher values of moisture content; 26.62±0.50g/100g in Tualang and 27.41±0.46g/100g in Gelam honey procured by Apisdorsata bees.⁴⁹ The cause of the higher moisture content may be unprocessed honey, collection at the time of rainy season,⁵³ high moisture leads to undesirable fermentation of the honey during storage caused by the action of osmotolerant yeasts, which results in the formation of C₂H₅OH (ethyl alcohol) and CO₂ (carbon dioxide). The alcohol can be further oxidized to acetic acid and water, which leads to a sour taste.^{54, 55} The lower moisture content observed in the rest of the samples may be attributed to the hotter weather and the decreased rainfall, especially from the beginning of the year to the middle of the year and as it contributes to its ability to resist the growth of micro-organisms, fermentation, and granulation during storage thus promoting a longer shelf life.^{36, 47, 56-58}

pH value

The parameter of pH is important during honey extraction and storage because pH influences its texture, stability and shelf life.^{49, 59} Honey is characteristically acidic in nature,it consists of organic acids, particularly gluconic, pyruvic, malic and citric acids; lactones; esters; and some inorganic ions, such as phosphate and chloride.^60-62 The obtained values of pH are 4.27 in the fresh honey and 4.10 in the old honey (Table 2), which were within the recommended limits (pH 3.42 to 6.10) for fresh honey.⁶³ It indicates whether the various processing conditions or 1 year of storage does not significantly affect the pH of honey. pH value is a useful criterion of possible microbial growth. The high acidity of honey is an indication of the fermentation of sugars present in the honey into organic acid, which is responsible for two important characteristics of honey i.e. flavor and stability against microbial spoilage.^{55, 64-66} The variation in pH of different honey samples are described to be due to the floristic composition and floral diversity of the regions.^{67, 68}

Acid Value and Total Soluble Solids (TSS)

The acid values were 15.00mgKoh/g and 7.62mgKoh/g and Total soluble solids 68.6^ob and 75.15^ob in freshly procured and old honey (Table 2), respectively.

Reducing Sugar

Sugars are the primary constituents of honey, and the key sugars are fructose and glucose, which is reducing or invert sugar. The total reducing sugar contents in the Pauttika honey samples were 20.59g/100g in the fresh honey and 27.49g/100g in old honey (Table 2), which was less than the limit ≥ 45 set by the CAC.⁵¹ The room temperature storage may increase the reducing sugar contents of honey because higher temperatures result in the evaporation of water, thus resulting in more concentrated honey.⁶¹

Hydroxyl methyl furfuraldehyde  (HMF) Content

HMF is used to determine the degree of deterioration of the honey and to indicate the purity and freshness of honey. The mean HMF value was4.13mg/100g and 11.39mg/100g in fresh and old honey respectively(Table 2), which were very less than 60mg/kg suggested by CAC[51]. M Rabiul Islam et al., 2017 have reported a similar finding of HMF (4.4±0.2 mg/100g) in fresh honey.⁴⁷ HMF is usually present in traces in fresh honey, but its levels tend to increase during processing and/or due to aging and also influenced by several other factors, such as pH, temperature, duration of the heat process, storage conditions and floral source; therefore, HMF levels provide an indication of overheating and poor storage conditions.^{66, 69}

Total Phenolic Content

Total phenolic content was 3mgGAE/g and 0.26mgGAE/g in the fresh and old honey respectively (Table 2), the finding of old honey was something similarto 0.8 mg/g of a study of Abdulaziz S. Alqarni et al., 2012.⁷⁰ Total phenolic content is a good criterion to determine the quality and curative properties of honey.⁷¹ Reportedly, polyphenols, flavonoids, and anthocyanins are the major bioactive compounds in foods and beverages that contribute significantly to the taste, texture, color, appearance, antioxidant activity, and functional properties.⁷² It may be beneficial for health and may give the therapeutic impact of chronic diseases.^{46, 73} The presence of phenolic acids and flavonoids in honey samples might act as a proton donator leading to the formation of acidified honey with low pH value.⁷⁴

Antioxidant Activity (% DPPH inhibition)

DPPH is a stable nitrogen-centered radical and has been widely used to test the free radical scavenging ability of various samples.⁶⁷ The percentage of DPPH scavenging activity of Pauttika honey was 82.51% and 60.77% in the fresh and old honey respectively (Table 2), which were similar to the previous studies; Bangladeshi honey 70.1±0.9mg/ml⁴⁷ and stored more than one-year results was 62.77± 24.18%.⁵⁸ This may be due to the presence of high amounts of polyphenols as well as other functional components in these honey types.⁴⁷

Vitamin C

Ascorbic acid is one of the non-enzymatic antioxidant substances present in honey.^{64, 75, 76} The ascorbic acid content of the fresh Pauttika honey was around 2mg/100g and 4mg/100g in old honey (Table 2), while a study by M Rabiul Islam et al., 2017 has analyzed the vitamin C content in 10 samples of honey (Apis dorsata), the mean value obtained was 107.3±10.8mg/kg.⁴⁷ The high content of ascorbic acid indicates a high antioxidant capacity of honey.⁷⁷

Minerals Content

Analysis of mineral content is an important index of possible environmental pollution, the presence of toxic content and a potential indicator of the geographical origin of honey. Mineral contents in the honey of the Pauttika honey are shown in Table 3. In this study, a total of four elements i.e. Ca, Fe, Mg, and Zn were quantified, and the obtained values of these minerals are Ca (2.93ppm), Mg (4.47ppm), Fe (0.34ppm), and Zn (4.47ppm). It is well known that mineral elements are involved in various physiological and metabolic processes, especially in bone formation, blood clotting, muscle contraction, and enzyme activity. Therefore, honey is popularly used as a good source of nutritional supplements.⁴⁷

Table 3: Minerals analysis of fresh Pauttika honeysample.

N: number of replicates

Sensory Evaluation of Honey

The data pertaining to sensory evaluation of honey fresh and old honey stored at room temperature (Table 4) showed that statistically higher scores (6.9) were given to honey at the fresh stage and the scores for sensory quality were increased during storage. The overall acceptance score in old honey was 7.4 which suggest the more acceptability of old honey than fresh.

Table 4: Sensory evaluation score (by 9 points hedonic rating scale) of fresh and old (one year stored at room temperature) Pauttika honey (Mean ± standard deviation values)

The color and appearance of fresh honey were liked moderately (score 7) which was less than the one-year-old Pauttika honey (liked very much score 7.2). The score of Consistency, flavor, and mouthfeel  were 7, 7.34, 7 in old and 7, 7.7, 7.8 in fresh honey, respectively. The higher sensory evaluation scores in old honey than fresh honey suggest that the acceptability of old honey was more than fresh honey. So the overall acceptance of one-year-old honey was more acceptable than the fresh honey on the hedonic rating scale.

Color (hunter color lab)

A different trendwas observed in the color hunter lab of honey after subjection to during the storage period, with respect to botanical origin (Table 5). In particular, L* values were decreased in Pauttika honey old sample afterthe passing of one year. Red components values (a*) and yellow components values (b*) were increased in Pauttika honey old sample afterthe passing of one year.

Table 5: Color (hunter color lab) test of fresh and old (one year stored at room temperature) Pauttika honeysample.

The analysis of the hunter color lab of Pauttika honey at the fresh and old honey whereas L* which shows lightness and darkness of the color whereas a* scale shows redness and greenness of the product and b* scale shows yellowness and blueness of the product. So, the values of L* scale in fresh honey showed darkness, which was less dark in comparison with old honey. The value of a* scale and b*scale of fresh honey were having less redness and yellowness in comparison of old honey respectively. Overall the color of the fresh honey sample was grayish, yellowish with less quantity of redness whereas in the old honey was more dark with yellowish and redness.

FTIR (Fourier-Transform Infrared Spectroscopy)

FTIR Spectrum is a technique to measure the wavelength of a sample and find out the peak value, characteristic bands of different functional groups. The value obtained in the Pauttika honey sample is mentioned in Table 6 & Fig 6.

Figure 6: FTIR Spectrum of Pauttika honey  Click here to View figure

Table 6: FTIR spectroscopy of old (one year stored at room temperature) Pauttika honey.

FTIR spectroscopy has shown the presence of different characteristic peak value with various functional compounds such as alcohol, carboxylic acid, alkyne, amine salt, alkane, imine/oxime, alkene, conjugated alkene, cyclic alkene, alkene, sulfate, phenol, sulfonate, sulfonamide, sulfuric acid, sulfone, fluoro compound, aromatic ester, alkyl aryl ether, primary alcohol, sulfoxide, amine, 1,2,4- trisubstituted, 1,3 disubstituted, halo compound, 1,4 disubstituted or 1,2,3,4- tetrasubstituted, 1,2,3 trisubstituted, 1,2 disubstituted that may be responsible for various medicinal properties.

Strength, Recommendation and Limitations of the Study

The naturally procured honey has been identified as Pauttika honey a specific kind described in Ayurveda is one of the strengths of present study. Pauttika honey has various medicinal properties; viz. hypolipidemic action, indicated in diabetes, etc. which can be further useful for the treatment of some kind of diseases. Characteristics of honey is influenced by a number of factors such as geographical origin, botanical (flower) sources of nectar, environmental and climatic conditions as well as it’s harvesting, processing and storage techniques, but researchers have taken only one sample of honey from one state. So, there can be further research on various samples of honey and honey bees procured from the different places in India and identifiation of  eight kind of honey bees and honey could be done as mention in Ayurveda texts. The experimental and clinical studies could be conducted on various kind of honey and their  properties as  mentioned in the Ayurvedic text.

FTIR spectroscopy has reported various functional groups. Further research on the analysis of sub-compounds of this honey may be conducted.

Conclusion

This study was the first attempt to standardize and validate Pauttika honey as mentioned in Ayurveda texts and as per modern parameters viz. physico-chemical analysis, organoleptic characters, hedonic scale, and hunter color lab, etc. The honey bee was identified as Apisdorsata and the Physico-chemical analysis of honey samples confirmed the good quality of honey according to the standards set by the Codex Alimentarius Commission. FTIR spectroscopy showed the presence of various functional compounds that may be responsible for various medicinal properties. The standard analysis of honey sample confirms that the procured honey was Pauttika types of honey among the eight kinds of honey as already mentioned in the ancient texts of Ayurveda. Analysis of fresh and old honey showed a difference in the organoleptic evaluation, hedonic scale, color and physico-chemical analysis which is consistent with the difference in properties of fresh and old honey as mentioned in Ayurveda. The overall acceptability of old honey was found better than fresh honey after getting the above analysis.

Acknowledgments

The author expresses her sincere gratitude to her supervisor and co-authors for their guidance and providing basic facilities during this study. Grateful thanks to Ida Tiwari (Professor), Department of Chemistry (Forensic Science), Satyaprakash Singh,Shbbir Raza Khan, Priya Aradhya Ekka,  Madhulika,  Aman, Imran Khan, Suraj to providing support. I acknowledge sincere thanks to Head of Department of Kriya Sharir, Department of Dairy Science and Food Technology, BHU and Division of Germplasm Conservation and Utilization, ICAR- National Bureau of Agricultural Insect Resources, Bangalore- 560024 Karnataka, India.

Funding Source

This study did not get funding from anywhere.

Conflict of Interest

The authors have no conflict of interest to declare.

References

1. Eteraf-Oskouei T, Najafi M. Traditional and modern uses of natural honey in human diseases: a review. Iran J Basic Med Sci. 2013;16(6):731–42.
2. Zumla A, Lulat A. Honey – A Remedy Rediscovered. J R Soc Med. 2018;82(7):384–5.
3. Bogdanov S. Honey in Medicine: A Review. Bee Prod Sci. 2017;1–28.
4. Kushwaha HCS, editor. Sutrasthana. In: Caraka Samhita. Reprint ed. Varanasi: Chaukhambha Orientalia, Varanasi; 2001. p. 445.
5. Susruta. Sutrasthana. In: Shastri KA, editor. Susruta Samhita. Reprint Ed. Varanasi: Chaukhambha Sanskrit Sansthan, Varanasi; 2010. p. 232,233,139.
6. Susruta. Sutrasthana. In: Sastri KA, editor. Susruta Samhita. Reprint ed. Varanasi: Chaukhambha Sanskrit Sansthan, Varanasi; 2010. p. 234.
7. Susruta. Sharirsthana. In: Shastri KA, editor. Susruta Samhita. Reprint. Varanasi: Chaukhambha Sanskrit Sansthan, Varanasi; 2010. p. 113.
8. Vagbhata. Uttarsthana. In: Gupta KA, editor. Astangahridyam. Reprint. Varanasi: Chaukhambha Prakashan; 2012. p. 9,12,13,47-48.
9. Vagbhata. Uttarsthana. In: Srikantha Murthy KR, editor. Astangsamgraha. Second. Varanasi: Chaukhambha Sanskrit Series Office; 2007.
10. Caraka. Sutrasthana. In: Kushwaha HCS, editor. Caraka Samhita. Reprint. Varanasi: Chaukhambha Orientalia, Varanasi; 2001. p. 445,447.
11. Susruta. Sutrasthana. In: Shastri KA, editor. Susruta Samhita. Reprint. Varanasi: Chaukhambha Sanskrit Sansthan, Varanasi; 2010. p. 233.
12. Caraka. Sutrasthana. In: Kushwaha HCS, editor. Caraka Samhita. Reprint. Varanasi: Chaukhambha Orientalia, Varanasi; 2001. p. 458.
13. Molan PC. The Antibacterial activity of Honey 2 . Variation in the potency of the antibacterial activity Variation in antibacterial activity. Bee World. 1992;73(2):59–76.
14. Bogdanov S. Antibacterial substances in honey. Swiss Bee Res Cent en www apis admin ch/english/pdf/BeeProducts/AntibacterialInternet\_e pdf. 1997;(8):1–10.
15. Molan PC. The Antibacterial Activity of Honey 1. The Nature of the Antibacterial Activity. 2001;5–28.
16. Erejuwa OO, Sulaiman SA, Wahab MSA. molecules Honey: A Novel Antioxidant. Molecules. 2012;17:4400–23.
17. Theunissen F, Grobler S, Gedalia I. The antifungal action of three South African honeys on Candida albicans. Apidologie. 2001;32:371–9.
18. Irish J, Carter DA, Shokohi T, Blair SE. Honey has an antifungal effect against Candida species.
19. Koc AN, Silici S, Ercal BD, Kasap F, Hörmet-Öz HT, Mavus-Buldu H. Antifungal Activity of Turkish Honey against Candida spp. and Trichosporon spp: an in vitro evaluation. Med Mycol. 2009 Nov 3;47(7):707–12.
20. Candiracci M, Citterio B, Diamantini G, Blasa M, Accorsi A, Piatti E. Honey Flavonoids, Natural Antifungal Agents Against Candida Albicans. Int J Food Prop. 2011;14:799–808.
21. Zhao X, Zhou Z, Han Y, Wang Z, Fan J, Xiao H. Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey. Microbiol Res. 2013 Nov 7;168(9):598–606.
22. Erejuwa OO, Nwobodo NN, Akpan JL, Okorie UA, Ezeonu CT, Ezeokpo BC, et al. Nigerian Honey Ameliorates Hyperglycemia and Dyslipidemia in Alloxan-Induced Diabetic Rats. Nutrients. 2016 Feb 24;8(3):95.
23. Sherlock O, Dolan A, Athman R, Power A, Gethin G, Cowman S, et al. Comparison of the antimicrobial activity of Ulmo honey from Chile and Manuka honey against methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. BMC Complement Altern Med. 2010 Sep 2;10.
24. Erejuwa OO, Sulaiman AS, Wahab SAM. Honey – A Novel Antidiabetic Agent. Int J Biol Sci. 2012;8(6):913–34.
25. Molan PC. The limitations of the methods of identifying the floral source of honeys. Bee World. 1998;79(2):59–68.
26. Murthy Srikantha KR. Madhu Varge. In: Bhavaprakasa of Bhavamishra. Reprint Ed. Chaukhambha Krishnadas Academy, Varanasi; 2008. p. 485–8.
27. Caraka. Sutrasthana. In: Kushwaha HCS, editor. Caraka Samhita. Reprint. Varanasi: Chaukhambha Orientalia, Varanasi; 2001. p. 446.
28. Ellis JD, Ellis A. African Honey Bee, Africanized Honey Bee, Killer Bee, Apis mellifera scutellata Lepeletier (Insecta: Hymenoptera: Apidae). 2009;1–7.
29. Jack CJ, Lucky A, Ellis JD. Giant Honey Bee Apis dorsata Fabricius ( Insecta : Hymenoptera : Apidae ) 1. 2019;1–6.
30. Kędzierska-Matysek M, Florek M, Wolanciuk A, Skałecki P. Effect of freezing and room temperatures storage for 18 months on quality of raw rapeseed honey (Brassica napus). J Food Sci Technol. 2016 Aug;53(8):3349–55.
31. Yilmaz H, Kufrevioglu I. Composition of Honeys Collected from Eastern and South-Eastern Anatolia and Effect of Storage on Hydroxymethylfurfural Content and Diastase Activity. Tuek J Agric. 2001;25:347–9.
32. Visquert M, Vargas M, Escriche I. Effect of postharvest storage conditions on the colour and freshness parameters of raw honey. 2013;
33. Castro-Vázquez L, Díaz-Maroto MC, González-Viñas MA, De La Fuente E, Pérez-Coello MS. Influence of storage conditions on chemical composition and sensory properties of citrus honey. J Agric Food Chem. 2008;56(6):1999–2006.
34. Semkiw P, Skowronek W, Skubida P, Rybak-chmielewska H, Szczęsna T. Changes Occurring in Honey During Ripening Under Controlled Conditions Based on Α-Amylase Activity , Acidity and 5-Hydroxymethylfurfural Content. J Apic Sci. 2010;54(1):55–64.
35. Sancho MT, Muniategui S, Huidobro JF, Lozano JS. Aging of Honey. J Agric Food Chem. 1992;40(1):134–8.
36. Moniruzzaman M, Sulaiman SA, Azlan SAM, Gan SH. Two-year variations of phenolics, flavonoids and antioxidant contents in acacia honey. Molecules. 2013 Nov 27;18(12):14694–710.
37. Droege S, Connor E, Gemmill-Herren B. Protocol to Detect and Monitor Pollinator communities Guidance for Practitioners. 2016. 64 p.
38. Official Methods of Analysis of AOAC International Association of Official Analytical Chemists. Vol. 15th. Arligton, Virginia, USA.; 1990.
39. Miller GL. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar GAIL. Anal Chem. 1959;31(3):426–8.
40. Keeney M, Bassette R. Detection of Intermediate Compounds in the Early Stages of Browning Reaction in Milk Products. J Dairy Sci. 1959;42(6):945–60.
41. Hinneburg I, Damien Dorman HJ, Hiltunen R. Antioxidant activities of extracts from selected culinary herbs and spices. Food Chem. 2006 Jul;97(1):122–9.
42. Mimica-Dukic N, Bugarin D, Grbovic S, Mitic-Culafic D, Vukovic-Gacic B, Orcic D, et al. Essential oil of myrtus communis l. As a potential antioxidant and antimutagenic agents. Molecules. 2010;15(4):2759–70.
43. Plummer TD. An Introduction to Practical Biochemistry. Biochem Educ. 1978;6(3).
44. Amerine MA, Pangborn RM, Roessler EB. Principles of sensory evaluation of food. Princ Sens Eval food. 1965;
45. Kisan M, Sangathan S. Indian Standard Guide for Sensory Evaluation of Foods, Part II: Methods and Evaluation Cards. New Delhi; 1997.
46. Samat S, Enchang FK, Hussein FN, Iryani W, Ismail W. Four-Week Consumption of Malaysian Honey Reduces Excess Weight Gain and Improves Obesity-Related Parameters in High Fat Diet Induced Obese Rats. 2017;2017.
47. Islam MR, Pervin T, Hossain H, Saha B, Hossain SJ. Physicochemical and Antioxidant Properties of Honeys from the Sundarbans Mangrove Forest of Bangladesh. Prev Nutr food Sci. 2017 Dec;22(4):335–44.
48. Fahim H, Dasti JI, Ali I, Ahmed S, Nadeem M. Physico-chemical analysis and antimicrobial potential of Apis dorsata, Apis mellifera and Ziziphus jujube honey samples from Pakistan. Asian Pac J Trop Biomed. 2014;4(8):633–41.
49. Peng Kek S, Ling Chin N, Aniza Yusof Y, Wei Tan S, Suan Chua L, Kek P. Classification of entomological origin of honey based on its physicochemical and antioxidant properties. Int J Food Prop. 2018;20(S3):S2723–S2738.
50. Yanniotis S, Skaltsi S, Karaburnioti S. Effect of moisture content on the viscosity of honey at different temperatures. J Food Eng. 2006 Feb 1;72(4):372–7.
51. Revised Codex Standard for Honey. Standards and Standard Methods. 2001.
52. Terrab A, Gonzalez AG, Diez MJ, Heredia FJ. Characterisation of Moroccan unifloral honeys using multivariate analysis. Eur Food Res Technol. 2003 Dec 1;218(1):88–95.
53. Pattamayutanon P, Angeli S, Thakeow P, Abraham J, Disayathanoowat T, Chantawannakul P. Biomedical Activity and Related Volatile Compounds of Thai Honeys from 3 Different Honeybee Species. J Food Sci. 2015;80(10):M2228–40.
54. Chirife J, Zamora MC, Motto A. The correlation between water activity and % moisture in honey: Fundamental aspects and application to Argentine honeys. J Food Eng. 2006 Feb;72(3):287–92.
55. Moniruzzaman M, Sulaiman SA, Khalil MI, Gan SH. Evaluation of physicochemical and antioxidant properties of sourwood and other Malaysian honeys: a comparison with manuka honey. Chem Cent J. 2013;7(138):1–12.
56. Singh N, Bath PK. Quality evaluation of different types of Indian honey. Food Chem. 1997 Jan;58(1–2):129–33.
57. Terrab A, González AG, Díez MJ, Heredia FJ. Characterisation of Moroccan unifloral honeys using multivariate analysis. Eur Food Res Technol. 2003;218(1):88–95.
58. Islam A, Khalil I, Islam N, Moniruzzaman M, Mottalib A, Sulaiman SA, et al. Physicochemical and antioxidant properties of Bangladeshi honeys stored for more than one year. BMC Complement Altern Med. 2012 Oct 8;12(177):1–10.
59. Peng Kek S, Ling Chin N, Aniza Yusof Y, Wei Tan S, Suan Chua L, Kek P. International Journal of Food Properties Classification of entomological origin of honey based on its physicochemical and antioxidant properties. Int J Food Prop. 2018;20(S3):S2723–S2738.
60. Echigo T, Takenaka T. Production of organic acids in honey by honeybees. J Agric Chem Soc Japan. 1974;48(4):225–30.
61. Khalil I, Islam A, Alam N, Gan SHUA, Sulaiman SA. Irradiation and evaporation enhance physicochemical characteristics, AEAC , FRAP , protein and proline contents of tualang honey. J Food Biochem. 2015;39:742–53.
62. Amssalu Bezabeh, Nuru Adgaba, Radloff S. &, Hepburn H. R. Physico-chemical methods for the characterisation of unifloral honeys: a review. Apidologie, Springer Verlag. 2004;35(1):71–81.
63. Khalil I, Moniruzzaman M, Boukraâ L, Benhanifia M, Islam A, Islam N, et al. Physicochemical and antioxidant properties of Algerian honey. Molecules. 2012 Sep 20;17(9):11199–215.
64. Bogdanov S, Jurendic T, Sieber R, Gallmann P. Honey for nutrition and health: A review. J Am Coll Nutr. 2008;27(6):677–89.
65. Bogdanov S. Honey Composition. Honey B. 2014;2019(Chapter 5):27–36.
66. Moniruzzaman M, Khalil MI, Sulaiman SA, Gan SH. Physicochemical and antioxidant properties of Malaysian honeys produced by Apis cerana, Apis dorsata and Apis mellifera. BMC Complement Altern Med. 2013 Feb 23;13(43):1–12.
67. Saxena S, Gautam S, Sharma A. Physical, biochemical and antioxidant properties of some Indian honeys. Food Chem. 2010 Jan;118(2):391–7.
68. Fahim H, Dasti JI, Ali I, Ahmed S, Nadeem M. Physico-chemical analysis and antimicrobial potential of Apis dorsata, Apis mellifera and Ziziphus jujube honey samples from Pakistan. Asian Pac J Trop Biomed. 2014 Aug;4(8):633–41.
69. Khalil MI, Sulaiman SA, Gan SH. High 5-hydroxymethylfurfural concentrations are found in Malaysian honey samples stored for more than one year. Food Chem Toxicol. 2010 Aug;48(8–9):2388–92.
70. Alqarni AS, Owayss AA, Mahmoud AA. Physicochemical characteristics, total phenols and pigments of national and international honeys in Saudi Arabia. Arab J Chem. 2012;9:114–20.
71. Al-Mamary M, Al-Meeri A, Al-Habori M. Antioxidant activities and total phenolics of different types of honey. Nutr Res. 2002 Sep;22(9):1041–7.
72. Cimpoiu C, Hosu A, Miclaus V, Puscas A. Determination of the floral origin of some Romanian honeys on the basis of physical and biochemical properties. Spectrochim Acta Part A Mol Biomol Spectrosc. 2013 Jan;100:149–54.
73. Baltrušaitytė V, Venskutonis PR, Čeksterytė V. Radical scavenging activity of different floral origin honey and beebread phenolic extracts. Food Chem. 2007 Jan;101(2):502–14.
74. Moussa A, Saad A, Noureddine D. How Honey Acts as an Antioxidant? Med Aromat Plants. 2012;1(1):1–8.
75. Alvarez-Suarez JM, Tulipani S, Romandini S, Bertoli E, Battino M. Contribution of honey in nutrition and human health: A review. Med J Nutrition Metab. 2010;3(1):15–23.
76. Board NH. Honey-health and therapeutic qualities. Natl Honey Board. 2002;1–28.
77. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 2005;91(3):571–7.