A comparative study on the role of Omani honey with various food supplements on diabetes and wound healing

2.1.1 Collection of samples

Mountain honey-MH (multifloral honey), cultivar honey-CH (Sidr honey), turmeric, lemon, garlic and ginger were collected from different places in the Sultanate of Oman, Muscat, Rustaq and Bahla. The respective honey samples were harvested in the month of early September 2017 and collected from local beekeepers and stored at room temperature in a dark place throughout the experimental period. Whereas food supplements used in the experiment were collected freshly time to time during the experiment time.

2.1.2 Preparation of honey with supplements

Twenty percent (20%) honey is prepared by mixing 20 g of honey in 80 ml of distilled water. The various food supplements (lemon, turmeric, garlic & ginger) extracts mixtures were prepared by mixing 1 ml of pure crude extract of the supplements in 100 ml of distilled water 1% (v/v).

2.1.3 Preparation of honey-ginger/turmeric extracts solution

Ginger and turmeric were washed with tap water and sliced into uniform pieces using sterile knife and dried in microwave at 40 °C for 48 h. The dried ginger and turmeric pieces were crushed using electric grinder to fine powder. Ginger and turmeric extracts were obtained by mixing 1 g of ginger/turmeric powder in 100 ml (1% W/V) of deionized water.

2.1.4 Preparation of garlic extract

Fresh garlic bulbs were cleaned and peeled, and homogenized using blender. The homogenized bulbs were squeezed using cheese cloth. The extract was prepared by diluting the concentrated garlic juice with distilled water (1 ml in 99 ml distilled water).

2.1.5 Preparation of lemon extract

The extract was prepared by diluting the pure lemon juice with distilled water (1 ml in 99 ml distilled water).

2.2.1 Total phenolic content (TPC)

The total phenolic content was assayed using the Folin-Ciocalteu method according to Socha et al. (2009) and Ferreira et al. (2009) with slight modifications. The honey solutions were prepared at a concentration of 8%. 0.5 ml of the stock solution was mixed with 0.3 ml of Folin-Ciocalteu reagent and followed by 2 ml of 15% sodium carbonate, the final volume was adjusted to 5 ml with distilled water and the mixture was incubated for 90 min. The absorbance of the mixture was measured at 765 nm against the blank. A standard curve of Gallic acid was prepared for quantification, using a concentration range between 8 and 40 mg/l and the results were expressed as mg Gallic acid/kg honey.

2.2.2 Total flavonoid content (TFC)

Total flavonoids content in honey was determined by a colorimetric method according to Zhishen et al. (1999). One ml of 0.2% honey was mixed with 4 ml of distilled water followed by 0.3 ml of 5% sodium nitrite. After 5 min, 0.3 ml of 10% aluminum chloride was added, six min later 2 ml of 1 M of sodium hydroxide was added and the final volume was increased to 10 ml. The mixture was shaken and absorbance was measured at 510 nm using a spectrophotometer. A calibration curve was made using a standard solution of catechin 20–100 mg/l. The results were presented as mg of catechin equivalents per kg of honey.

2.3.1 DPPH free radical-scavenging

The antioxidant characteristics of mountain and cultivar honey samples were examined by determining the free radical-scavenging activity of the DPPH radical based on the method Ferreira et al. (2009). In brief, honey samples were dissolved in distilled water at various concentrations (50%, 75%, and 100%) from 50 to 100 mg/ml. 0.5 ml of honey was mixed with 2.7 ml of methanolic solution containing DPPH radicals (0.0039 g/ml). The mixture was vigorously shaken and left to stand for 15 min in dark. The decrease of the DPPH radical was determined by measuring the absorbance of mixture at 517 nm against blank. The blank consisted of a honey solution (1.5 ml) at the same concentration values previously described and 3.5 ml of methanol. The negative control consisted of 3.5 ml of methanol with 1.5 ml of the DPPḢ solution.

Ascorbic acid was used as a standard. The radical scavenging activity (RSA) was calculated as the percentage of DPPH discoloration using the following equation:$$\%\text{RSA}=\left[\left({\text{A}}_{\text{DPPH}}-{\text{A}}_{\text{s}}\right)\right]/{\text{A}}_{\text{DPPH}}\times 100$$where A_s is the absorbance of the solution when the sample extract is added at a particular level and A_DPPH is the absorbance of the DPPH solution.

2.3.2 Total antioxidant capacity

Three hundred ul (0.3 ml) of honey (honey + 20 ml Methanol) was mixed with 2.7 ml of Phosphomolybdenum reagent (0.335 g of sodium phosphate and 0.078 g of ammonium molybdate in 3.3 ml sulphuric acid) in test tube. The samples in a test tube were kept in a water bath at 95 °C for 90 min. The blank was prepared by mixing 0.3 ml of methanol without honey. The absorbance of the samples was measured at 695 nm using UV–Vis spectrophotometer. Ascorbic acid was used as control. Different concentrations were used for honey as well as ascorbic acid: −100%, 75% and 50%. The total antioxidant capacity was estimated (Shumaila et al., 2013) using the following formula:

Total antioxidant capacity (%) = [(Abs of control) - (Abs of sample)] / (Abs of control) X 100

2.4.1 Induction of diabetes

The Healthy male experimental mice is segregated as different groups were fasted overnight (12–14 h) and their weight and blood glucose level was noted. Mice were then made diabetic by a single intraperitoneal injection (150 mg/kg body weight) of alloxan monohydrate. Two days after alloxan injection, plasma blood glucose level of each animal was determined by taking the blood from the tail of mice and recorded by glucometer select plus model.

2.4.2 Experimental design

The healthy male mice were divided into 8 groups for the estimation of blood glucose level with 5 animals in each group as follows. Group 1: negative control (Diabetic mice, no treatment), group 2: positive control (treated with glibenclamide-5 mg/ml), group 3: normal mice, group 4: treated with Mountain honey (MH), group 5: treated with MH + G, group 6: treated with MH + L, group 7: treated with MH + G* (mountain honey with ginger) and group 8: treated with MH + T. The same experimental regimen was maintained for cultivar honey (CH). All experimental mice were received treatment after 2 days of alloxan injection except normal mice (nondiabetic) and diabetic control (positive & negative) groups. The dosages were administered to the mice orally thrice a week along with regular mice feed and water. The blood sugar levels and body weights were measured prior to the treatment and after three weeks of treatment using glucometer device and weighing balance respectively.

3.4.1 Effect of honey in combination with food supplements on blood sugar levels in diabetic mice

From the Table 1(a)–(d) it is noticed that the consumption of honey alone and with supplements has shown diverse results against blood sugar level in diabetic mice. The Alloxan administered mice with sugar level 110 mg/dl and above was considered as diabetic mice. It was reported that honey composed of fructose at high concentration (Fasanmade and Alabi, 2008) as well as glucose. But in this study it was noticed that the cultivar honey reduced the blood glucose level from 154.6 ± 33.84 to 146 ± 13.00 (↓8.6 ± 20.84) than the mountain honey 134 ± 12.94 to 133.6 ± 18.09 (↓0.4 ± 5.15).

Though Honey contains fructose and glucose, the glycemic index is lower than refined sugar thereby the impact of honey on diabetics is lesser than refined sugar. Several reports suggests that fructose in honey may moderate the hypoglycemic effect of honey (Erejuwa et al., 2012). Also the honey with relatively high concentration of fructose has a lower glycemic index (GI) than other types of honey (Ischayek and Kern, 2006). In addition, in a comparison between honey in combination with supplements both MH from 143.8 ± 24.74 to 122.4 ± 11.82 (↓21 ± 12.92) and CH from 121.6 ± 19.50 to 116.6 ± 10.11 (↓5 ± 9.39) with turmeric showed the greatest reduction in the blood glucose level than other supplement combinations.

Turmeric is known to have many health benefits, and turmeric is one of the five medicinal plants used effectively as a single drug (Vuthipongse, 1986; Sithicharoenchai, 1986; Suppasil-Nanakorn, 1993). Turmeric alone is found to reduce the blood sugar from 135.3 ± 13.31 to 118.5 ± 24.74 (↓16.8 ± 11.43), lemon from 153.6 ± 5.13 to 147.6 ± 28.09 (↓9 ± 22.96) was also shown reduction in blood sugar level in diabetic mice. But lemon in combination with CH has shown increasing the blood sugar (↑20 ± 13.87), while lemon with MH the reduction (↓20 ± 2.07) of blood sugar level in diabetic mice was observed. From the overall observations MH in combination with different supplements studied was found to be more effective than CH treatment groups.

These findings clearly suggest that natural mountain honey is safer than cultivar honey from a diabetic point of view. Since, the glycemic index (GI) of the honey which contains more fructose (MH) have less GI than the honey (CH) which is having less fructose were GI will be high. Several reports revealed that lower GI foods are not affecting and rising blood sugar levels in type II diabetics (Bobiş et al., 2018). The positive control glibenclamide drug was used and showed a decline (↓3.7 ± 13.26) in blood glucose level in diabetic mice. While in normal (↑9.4 ± 14.59) control (not diabetic; normal mice) and negative (↑17.7 ± 8.00) control (diabetic; no treatment), the blood sugar level was elevated.

3.4.2 Effect of honey in combination with food supplements on body weight in diabetic mice

Results showed in Table 2(a)–(c) shows the effect of honey in combination with additives on the body weight in diabetic mice. Overall MH (mean difference ↑2.66 ± 1.374 g) alone and in combination with supplements has resulted in progressive increase in body weight of all the treated groups of diabetic mice similar to the standard drug treated mice (↑3.22 ± 0.360 g).

However CH alone shown the (18 ± 2.107 to 10.3 ± 2.517) mean difference (↓7.7 ± 0.410 g) and with supplement formulations has reduced the body weight in all treated diabetic mice. This was the major difference found and noticed among the two types of honey and this might be based on glycemic index. As mentioned earlier under antidiabetic study CH contains low concentration of fructose resulted in high glycemic index (Nemoseck et al., 2011). It was suggested that the honey which has a high glycemic index is assumed to increase the body weight, in controversy the results of Chepulis and Starkey (2007) reported that honey fed rats had significantly lowered the body weight, adiposity etc.

From the current results we noticed that the uptake of MH is better than the CH in gaining weight during diabetic condition. The MH seems to possess more of natural sugars than the CH leads to the diabetic mice gaining weight than losing with MH treatment. In addition, supplements alone also reduced the body weight of the mice. A great reduction of weight in diabetics was noticed with turmeric treatment from 28.1 ± 3.381 to 10 ± 0.902 mean difference (↓18.1 ± 2.479 gms) followed by garlic (↓15.3 ± 1.510), ginger (↓12 ± 0.662) and lemon (↓4.5 ± 0.146 gms). As seen in Table 2(a)–(c) the synergistic influence of honey with different food supplements does not influence the body weight during diabetes, but the individual supplements showed reduction of body weight in diabetics.

The supplement Turmeric showed great impact on blood sugar and in relation with body weight. This might be due to the presence of active ingredients in the turmeric called curcumin and curcuminoids. This evidences that even MH alone (↑2.66 ± 1.374 gms) and in combination with lemon (lemon ↑3.04 ± 0.087 gms) increased the body weight significantly. In comparison the synergistic effect with turmeric is very low (↑1.42 ± 0.239 gms), this suggests that turmeric in general causes weight reduction in individuals.