Abstract

The study was carried out to determine the therapeutic potential of commercial honey by ascertaining their antioxidant activity of four brands of (OH1, BH2, LH3 and LS4) commercial honey samples purchased from supermarkets within Choba community in Rivers State, Nigeria. The study was carried out from October 2017 to January 2018. Analysis of phenolic and flavonoids content were carried out by High Performance Liquid Chromatography (HPLC). For phenolic Florisil Column, mobile phase Toluene: cyclohexane: acetone (60:30:10) v/v, and Fluorescence detector were used with working standards of 0- 0.8 at wavelength of 765nm. Flavonoids were also analyzed by HPLC using mobile phase of 20: 10: 5 v/v and wave length of 506 nm. Results obtained from the study showed that % Vitamin E of honey sample was 0.098, 0.099, 0.105 and 0.121 while % vitamin C was highest in LS4 (0.535). % carotenoids were 0.046, 0.041, and 0.045. The highest percentage of carotenoids was in sample BH2. % proline values were 0.047, 0.067 and 0.045 particularly in two samples (LH3 & LS4). Forty-five phenolic acid of total phenolic contents obtained were 8.235, 9.632, 8.498 and 11.507 ppm with the highest value in sample LS4. Total flavonoid values obtained were 51.327, 35.687, 38.288 and 29.927 ppm. The study revealed that the high level of phenolic and flavonoid contents showed antioxidant capacity in the samples studied which were correlated to total phenol, total flavonoid content, vitamin C and E.

1. Introduction

Honey is a natural product that is referred to as a concentrated solution because of its unique composition. The composition of honey varies from one geographical location to another ¹. Based on the unique composition and medicinal properties, such as antimicrobial, bacteriostatic, anti-inflammatory, antioxidant, radical scavenging activity, antiviral and other properties, made it possible for honey to be ideal for some medicinal purposes and a natural food source ^{2, 3}. The floral origin of any honey determines the composition and content of the honey. Honey has been used for anti-inflammatory, antimicrobial activity and topical application. Antioxidant effect in honey is initiated by flavonoids, phenolic acids, enzymes, ascorbic, peroxidase, carotenoids, protein and other constituents. Antioxidant activity has to do with the chemical composition of the honey which is associated with the origin of the honey ^{4, 5}. Phenolic compounds and flavonoids are found in honey and they both possess antioxidant activity which depends on floral origin and the colour of the honey. Flavonoids can be classified into various sub groups such as flavonols, flavanones, flavones, anthocyanidins and isoflavones ^{6, 7}. Some studies have shown that the dark coloured honey have higher phenolic content and antioxidant activity than the light coloured honey ^{8, 9}. The colour of honey is associated with the phenolic and other compounds found in honey that come primarily from plants where the honey is been harvested. Flavonoids in honey have a lot of components that are responsible for the taste, aroma and antioxidant activity. Based on all these medical importance of honey most people visit the open markets and supermarkets to buy honey that is labeled natural or pure. The authenticity and originality of the honey cannot be ascertained by the label and physical appearance alone. Are these honey really natural product and contain antioxidant activity? The study was carried out to determine the therapeutic potential of commercial honey by ascertaining their antioxidant activity.

Flavonoid content was determined by High Performance Liquid Chromatography (HPLC) using Water 61/626 which was made up of Florisil Column, mobile phase; toluene: cyclohexane: acetone (20:10:5) v/v, and Fluorescence detector. The working standards were 0 - 0.4 ppm at wavelength of 506 nm. Honey samples for flavonoid analysis were extracted by adding 20 ml of methanol into 2 g of honey in an extraction tube and shaken for 25 minutes and allowed to stand for 1 hour. Aluminum trichloride (0.2 ml of 40%) in methanol was added followed by a drop of acetic acid (CH₃COOH) which was diluted with methanol to 250 ml. It was shaken for 15 minutes and allowed to stand for 45 minutes. The supernatants were transferred to HPLC auto analyzer, absorbance was read at 506nm ¹⁰.

Phenolic compounds were also determined by using the method of flavonoid analysis with the slight modification. Florisil Column, mobile phase: toluene: cyclohexane: acetone (60:30:10) v/v, and Fluorescence detector were used. Briefly, 5 g of each honey sample was weighed into a 250 ml digestion tube and 140 ml of 80% ethanol plus 60 ml of ultrapure water was added. Ten (10 mL) milliliter of the solution was transferred to a set of test tube and Na₂CO₃ (0.8 ml of 40% ) solution were added. The final volume was made up to 250 ml with ultrapure water. The solution was shaken vigorously for 10 minutes and allowed to stand for 4 hours. The samples extracts were transferred to a set of HPLC auto analyzer cups and analyzed on Water 616/626 at wavelength of 765nm.

The proline content in the honey samples was determined by the method, which was established by the International Honey Commission (IHC) ¹². Briefly, 5g of honey was transferred in to a beaker and dissolved in 50mL water. The solution was quantitatively transferred to a 100mL volumetric flask and further diluted to 100mL with distilled water. Approximately 0.5mL of the sample solution was transferred to a tube, while 0.5mL of water (blank test) was transferred to a second tube and 0.5mL of proline standard solution was dispensed into another set of 3 tubes. To each tube, approximately 1mL of formic acid and 1mL of ninhydrin solution were added. The tubes were capped carefully and shaken vigorously for 15min and then placed in a boiling water bath for 15min and immersed below the level of the solution. The tubes were further transferred to another water bath and incubated at 70°C for 10min. Approximately 5mL of the 2-propanol water solution was added to each tube followed by immediate capping. The tubes were left to cool for approximately 45min after removal from the 70°C water bath and the absorbance values were measured at 510nm.

2. Results

The antioxidant activity of four honey samples from Choba community in Rivers State Nigeria was determined. The honey samples were analyzed for flavonoid, phenolic and proline content. Thirty-one flavonoids were detected from five subgroups of flavonoids. Results obtained from flavonoids analysis of the samples are detailed Table 1. The most abundant flavonoids were Genistein from the isoflavones subgroup. The values obtained were [OH1 (9.5585 g 100g^{- 1}), BH2 (5.0706 g 100g^{- 1}), LH3 (7.8436 g 100g^{- 1}) and LS4 (4.2499 g 100g^{- 1})]. Glycetin was next to Genistein from the isoflavones subgroup. Among the flavanones subgroup, Nanirutin was the highest in occurrence with values ranging from 0.167 to 1.118 g 100g^{- 1}. This was followed by hesperidim with the values of 0.403, 0.510, 0.765 and 0.865 g 100g^{- 1}g, respectively. The least in occurrence among the flavonoids subgroups are flavones (Rhoifolin, Tangeretin, Nobiletin, Apigenin); Isoflavones (Daidzein); flavan-3-ols (Thearubigins) and flavanones (Eriodictyol, Naringerin, Raxifolin). No strong correlation was found between the honey samples for individual flavonoid content. Total flavonoid contents obtained for the honey samples were OH1 (51.327 ppm), BH2 (35.687 ppm), LH3 (38.288 ppm) and LS4 (29.927 ppm). The highest flavonoid content was obtained in OH1 honey and the least was in LS4. OH1 honey is a dark coloured honey which is manufactured and bottled in Imo state Nigeria, while LS4 honey being the foreign and not produced in Nigeria is a light coloured honey.

Forty-four phenolic acids were detected in the honey samples. The most abundant phenolic acids were as follows syringic acid, 4-hyroxybenzoic acid, gallic acid, vanillic acid, mandelic acid, homogentisic acid, cinnamic acid, phenylpropanoic acid, caffeic acid, ferulic acid and rosmarinic acid as mentioned in Table 2. Total phenol content for the honey samples was OH1 (8.235 ppm), BH2 (9.632 ppm), LH3 (8.498 ppm) and LS4 (11.07 ppm). Total flavonoid content of the honey samples was higher than total phenolic content. Honey sample LS4 with the least total flavonoid content had the highest phenolic content. Total phenolic content did not vary between honey samples OH1, BH2 and LH3.

The highest phenolic acid was gallic (0.143-1.277 g 100g^-1) followed by castarinol C3, which ranged from 0.221- 1.069 g 100g^{- 1}. The highest castarinol C3 content was observed in LS4 (1.069 g 100g^{- 1}) honey sample. This was followed by ethlycaffeati which had the highest in SH1 sample with acid content of 0.795 g 100g^{- 1}. Other values obtained were 0.051and 0.385 g 100g^{- 1} for BH2 and LH3 respectively. Cinnamic acid also had a notable value of 0.048 g 100g^{- 1} (OH1), 0.838 g 100g^{- 1} (BH2), 1.018 g 100g^{- 1}g (LH3) and 0.726 g 100g^{- 1} (LS4). LS4 had the highest phenolic acid also in gallic acid (1.227 g 100g^{- 1}) and phenyloacetic (0.535 g 100g^{- 1}). Valnilic acid ranged from 0.339 – 0.489 g 100g^{- 1}, benzoic acid ranged from 0.077 -0.111 g 100g^{- 1} while verasonic acid ranged from 0.492 – 0.646 g 100g^{- 1} in the honey samples studied. Significant difference was observed between total phenolic and flavonoid (Figure 1), this shows that flavonoid content were more than phenolic content in the present study.

The proline content, vitamin C, Vitamin E and Carotenoids for the honey samples was OH1 (0.047, 0.415, 0.098, 0.046 %); BH2 (0.067, 0.421, 0.099, 0.041 %); LH3 (0.045, 0.449, 0.105, 0.045) and LS4 (0.045, 0.535, 0.121, 0.045%) as shown in Figure 2 below. Proline content was highest in BH2 honey, no strong correlation variance was observed among carotenoids and proline. Significant difference was observed between vitamin C and E.

3. Discussion

A study carried out on 5 monofloral honey from Bangladesh characterized the phenolic acids, flavonoids and antioxidant properties. From their study, five different phenolic acids were identified, and the most abundant was caffeic acid, benzoic acid, gallic acid, chlorogenic acid and the least was trans-cinnamic acid ¹². In our study, the most abundant phenolic acid was gallic acid, Castarinol C3, ethylcaffeati, cinnamic acid, phenyloacetic, valnilic and verasonic acid. The least in occurrence in our study was Cyanidin glucose, Cyanidin coumaroyl glucose, castarinol C2 and C4, singlic acid and custisssin. Our study was in conformity with the study for some of the phenolic acids ¹². Differences observed may be attributed to the floral origin, vegetation, seasonal variation, the plants and nectar the bees feed on. To an extend storage method, processing and handling method of the honey may be responsible for the slight differences observed ¹⁴. Studies have shown that phenolic are very important in honey and they tend to have higher level of antioxidant activity when compared to vitamin C and E ¹⁴. Phenolic content in our study was lower than those obtained by Buba ¹⁴ with total polyphenol and vitamin C contents values of 65.31 ± 19.50 mg Gallic Acid Equivalent (GAE) and 21.15 ± 3.99 mg 100g^{- 1}, respectively. The present study was not in conformity with findings on Tualang honey ¹⁵. Their study obtained highest phenolic compound of (352.73 ± 0.81 mg gallic acid kg^-1) and flavonoid (65.65 ± 0.74 mg catechin g 100g^{- 1} kg) while the present study had the highest concentration for phenolic acid of 11.507 ppm for total phenolic content and 51. 327 ppm was highest total flavonoid. In Nigeria, the antioxidant properties of two stingless bee was studied, phenol content was (527.41 ± 3.60 mg kg^-1) and ascorbic acid (161.69 ± 6.70 mg kg^-1) ¹⁶. Several correlations were noted among some of the parameters of the honey samples. Other studies showed that total flavonoid content of the honey samples ranged from 53.81 ± 4.12 rutin equivalents kg^-1 to 549.05 ± 9.74 mg rutin equivalents kg^-1 while total phenolic content in both solvents ranged from 357.14±3.57 mg gallic acid equivalents kg^–1 to 520.83±4.49 mg gallic acid equivalents kg^{–1 17}.

Results obtained for flavonoid in Bangladesh honey showed that kaempferol, and catechin were most abundant, followed by myricetin and the least was naringenin ¹². In our study, most abundant flavonoid was genistein, glycetin, nanirutin, resperidim, iuteolin, epicatechin, catechin, epicatechin gallate and epigallocatechin gallate. The least in occurrence was rhoifolin, tangeretin, nobiletin, apigenin, daidzein, thearubigins, eriodictyol, naringerin and raxifolin, which correlated with some of the flavonoid in their study. A study on seven Korean honey showed that total phenolic content had a range of 5.27 to 29.17 mg of gallic acid equivalents^-1g of dry fraction. They observed high correlation coefficients between total phenolic contents and the antioxidant activities. This confirms that phenolic were mostly responsible for high level of antioxidant observed ¹⁸.

Results obtained for vitamin E in the present study was 0.098, 0.099, 0.105 and 0.121% for samples OH1, BH2, LH3 and LS4, while % vitamin C was ranged from 0.415 – 0.535 % for the honey samples respectively. Carotenoids result was 0.046, 0.041, and 0.045 (%). The highest percentage of carotenoids was in sample BH2. The proline values were 0.047, 0.067 and 0.045 %. Our result is in agreement with the result obtained by Wasagu ¹⁹ with antioxidant vitamin C and E content of two honey samples was 2.22± 0.10and 0.28± 0.03(mg ^-1 dl), (for light amber), 2.61± 0.11 and 0.26± 0.02 (for dark amber), respectively. Vitamin C was higher in their study than vitamin A. This is in agreement with the present study as presented in Figure 2, where dark honey samples had higher content of these vitamins than the light coloured honey. The differences observed among the different honey samples could be associated with the climatic conditions and vegetation in that particular region. Different nectar where the bees forage can also be responsible for differences observed between our study and those obtained by other researchers ^{20, 21, 22}. Proline indicates the ripeness of honey; high proline shows that the honey was matured and ripe before harvesting. When the proline content in honey is high, it reduces adulteration. Researchers attributed the reduced proline content to long storage of the honey ²³. Phenolic, flavonoids, proline, vitamins etc. all contribute to the antioxidant ability of honey in their own capacity. Studies have shown that when these components are separated from honey and tested in in vitro systems, only a portion of the total antioxidant activity of honey is produced. This further explains the fact antioxidant in honey occurs due to combined effect of all antioxidant components ²⁴. For honey to have the required antioxidant activity it must be authentic. Few commercial honeys from Nigeria are authentic based on the present study and previous studies by Agbagwa ^{25, 26} on the microbiological and physicochemical assessment of local and foreign honey sold in Nigeria. The result from that study showed that the moisture content had a range of 16.75 - 31.20 mg^-1 L for local honey while foreign samples ranged from 16.71 - 30.5 mg^-1 L. Lower moisture content was observed in the foreign samples and those from research institutes than some of the commercial honey. Differences were observed in the physicochemical parameters of both honey types, this difference rely solely on the region and floral difference, including other factors ²⁷. Honey with high moisture content has the ability to ferment and also create a good environment for microbial growth. According to Codex Alimentarius ²⁸ and EU ²⁹ Standard of honey samples, states that the moisture content in honey is a maximum of 21%.

4. Conclusion

The study therefor concludes that high level phenolic and flavonoid content was observed in commercial honey to an extent. This confirms the antioxidant capacity of the samples studied which were correlated to total phenol, total flavonoid content, vitamin C and E. To an extent the commercially available honey in the supermarkets are authentic and possess antioxidant potential.

References