Ajay Dongare*, Akshay Jagtap, Rajeshwari Bhise, Amir Shaikh, Suvarna Ingale, Sunil Kakad
SCES’s Indira College of Pharmacy, Pune, M.S. India
*Address for correspondence
SCES’s Indira College of Pharmacy, 89/2A, Tathawade, Pune, M.S. India
Objective: To evaluate honey samples with respect to their physicochemical properties and anti-inflammatory activity. Materials and Methods: Different samples of honey namely Saffron honey, Chambal honey, Ajwain honey and Tulsi honey were evaluated for Ash content, pH, Moisture content and anti-inflammatory activity. Results and discussion: Anti-inflammatory activity of honey samples with different concentrations (30, 40, 50, 60 mg/ml) were compared using HRBC membrane stabilization method. Honey samples namely Tulsi honey, Saffron honey, Ajwain honey and Chambal honey showed maximum protection of 94.12%, 85.28%, 79.36% and 74.35% respectively at 60 mg/ml concentration. The results revealed that as with increase in concentrations of honey, membrane stabilizing activity also increases significantly. Tulsi honey gave highest ash content of 0.54±0.25 gm/100gm. Maximum pH of 4.8±0.36 was shown by chambal honey while highest acidity was seen in tulsi honey (3.8±0.43). Conclusion: This study demonstrated that honey samples possess significant anti-inflammatory activity. Thus it can be concluded that, physicochemical properties, anti-inflammatory activity and quality of honey varied depending on the botanical origins, storage conditions and handling.
Keywords: Honey, anti-inflammatory activity, membrane stabilization
Humans are known to use honey since ancient times, nearly 4000 years ago (Adebolu, 2005). It is being used in various food products and beverages. Honey is also being used to treat various diseases along with the usage in foods and beverages and is being considered as a natural cure for a wide variety of diseases. Most ancient populations, including the Greeks, Chinese, Egyptians, Romans, Mayans, and Babylonians, consumed honey for its nutritional value and for the medicinal properties (Ashrafi et al., 2005). Honey has nutritional, cosmetic, therapeutic, and industrial values and it is the only insect-derived natural product (Bansal et al., 2005). At least 200 components are present in honey which mainly includes water and carbohydrates, along with proteins, flavonoids, minerals, vitamins, free amino acids, free enzymes, organic acid, phenolic acid and other phytochemicals (Terrab et al., 2003). Honey does not require refrigeration because it never get spoiled, and it can also be stored unopened at room temperature in a dry place.
The pH value of honey is around 3.9 and water activity (WA) of honey ranges between 0.56 to 0.62 (Hassapidou et al., 2006; Babacan S and Rand AG., 2007). Since honey has high level of fructose it was utilized as a natural sweetener from ancient period and it has 25% more sweetness than tablet sugar (Babacan and Rand, 2007; Pataca et al., 2007). Honey was known to possess various pharmacological properties viz anti-inflammatory, anti-bacterial, antioxidant etc. (Martos et al., 2000). For treatment of various infections, honey is used topically since earlier period (Benhanifia et al., 2011). Honey heals the wound, burns and pyorrhea or ulatrophia effectively (Cooper and Gray, 2012). Benhanifia et al demonstrated that number of inflammatory cells present in burned tissue and in full thickness wounds are reduced by using honey histologically and this effect was due to non-sugar component of honey (Benhanifia et al., 2011). Though, the appropriate mechanism of anti-inflammatory action of honey has remained unknown, honey inhibits the formation of prostaglandin, which is often responsible for the characteristics associated with inflammation such as heat, itchiness, swelling and pain (Kassim et al., 2010).
Now a day with the boon of using herbal medicine for the treatment of various ailments, use of honey as medicine as well as vehicle has become very common and market is flooded with various brands of honey. However, the composition, physicochemical characteristics and thereby efficacy of honey is known to vary according to geographical location and plant source. Hence, the present study was planned to investigate the physicochemical properties and anti-inflammatory activity of various honey samples namely Tulsi honey, Saffron honey, Ajwain honey and Chambal honey obtained from different plant source.
Materials and methods
Different samples of honey namely Saffron honey, Chambal honey, Ajwain honey and Tulsi honey were obtained / procured from Central bee research institute, Pune. All samples of honey were stored at room temperature. All required Chemicals of analytical grade were used.
Evaluation of physicochemical properties
Different samples of honey were examined and evaluated for its variations in potency and quality. Ash content, pH and Moisture content were determined.
According to methods of Association of Analytical Communities (AOAC), 1999, the ash content was determined; 5g of different samples of honey were placed in 5 different combustion pots. For prevention of honey foaming pots required preheating to darkness with a gas flame. Then with the help of burning muffle, the samples were incinerated at high temperature (550°C) for 5 hours followed by cooling the different ash samples at room temperature. Obtained ash was accurately weighed (Sohaimy et al., 2015).
For measuring pH of different honey samples a 211 microprocessor pH meter was used.
For measuring moisture content of different samples of honey Karl Fischer titrator (Matic D, Veego), was used.
Anti-inflammatory activity by HRBC membrane stabilization method
Collection of human erythrocyte suspension
The heparinized vacutainer was used to collect the blood sample from a healthy volunteer who had not taken any NSAIDs for 2 weeks prior to the experiment. The equal volume of 0.42% sodium chloride in water, 0.8% sodium citrate, 2% dextrose and 0.05% citric acid were mixed with the collected blood sample. The blood was centrifuged at 3000 rpm and isosaline (0.85%, pH 7.2) was used to wash the packed cells. Finally 10% v/v suspension was prepared by using isosaline.
Hypotonic solution-induced hemolysis
The HRBC membrane stabilization method was used to determine the anti-inflammatory activity of different honey samples. The assay mixtures were prepared which contained 1ml of phosphate buffer (0.15M, pH 7.4), 2ml of hyposaline (0.36%), 0.5ml of HRBC suspension and different concentrations (30, 40, 50, 60 mg/ml) of each honey sample. In blood control the honey was omitted. All assay mixtures centrifuged at 3000 rpm and were kept for 30 min at room temperature. The hemoglobin content was estimated at 560 nm using UV spectrophotometer. For calculating the percentage of HRBC membrane stabilization, following formula was used.
Percent protection = (1-ODt) / ODc x 100
ODt = absorbance of test sample,
ODc = absorbance of control/reference
Data was expressed as mean ± SEM. The data was analyzed by Student t test at p ≤ 0.01 for statistical significance.
Results and discussion
Various honey samples which are procured from central bee research institute, Pune were evaluated for their ash content, pH, moisture content, and anti-inflammatory activity.
The quality of many food materials depends on the concentration and type of minerals they contain, including their taste, appearance, texture and stability. It is important to know the mineral content of various ingredients as it may change the physicochemical properties of materials. The minerals in honey give a higher nutritional value than found in refined sugars or syrups (White, 1978).
Ash content of different honey samples was determined and results were depicted in figure 1. Maximum ash content was seen in tulsi honey (0.54±0.25 gm/100gm) while lowest was observed in chambal honey (0.22±0.43 gm/100gm).
Figure 1. Ash content of various samples of honey: (Mean ± S.D for n=3)
Fermentation of sugars into organic acid correlates high acidity of honey. This is responsible for flavor and stability of honey (Bogdanov et al., 2003). From figure 2, it can be seen that maximum pH of 4.8±0.36 was shown by chambal honey while highest acidity was seen in tulsi honey (3.8±0.43). Found results are in accordance with Codex Almentariou Commission (2001), which has given acceptable pH range of honey in between 3.40 to 6.10. Variation of different acid, floral difference and minerals present in the honey might be responsible for different pH of honey.
Figure 2. pH of various samples of honey: (Mean ± S.D for n=3)
For determination of quality, stability and spoilage resistance of honey against yeast fermentation, moisture content of honey is one of the limiting factor. Probability of honey fermentation during storage is higher if moisture content is higher. Lower moisture content (<20%) increases honey’s shelf life (Codex Alimentations, 2001). Lower moisture content was seen in tulsi honey (15.22±0.47 gm/100gm) while higher moisture content of 19.42±0.12 gm/100gm was shown by chambal honey (Figure 3).
Figure 3. Moisture content of various samples of honey: (Mean ± S.D for n=3)
Membrane stabilization activity
In-vitro anti-inflammatory activity of different samples of honey was using membrane stabilization method as erythrocyte membrane and lysosomal membrane is analogous to each other. By preventing the release of lysosomal constituents of activated neutrophil, stabilization of lysosomal membrane can be achieved. This is required in limiting the inflammatory response. Various disorders were produced by lysosomal enzymes which are released during inflammation. Acute and chronic inflammation is related with extra cellular activity of these enzymes (Leelaprakash and Mohan-Dass, 2011; Sadique et al., 1989).
The results of HRBC membrane Stabilization revealed anti-inflammatory activity to various samples of honey as depicted in figure 4A, 4B, 4C, 4D and 5. It was observed that as concentration of honey increases the membrane stabilization activity also increases. Different honey samples at a concentration of 30–60 mg/ml were evaluated. Honey samples namely Tulsi honey, Saffron honey, Ajwain honey and Chambal honey showed maximum protection of 94.12%, 85.28%, 79.36% and 74.35% respectively at 60 mg/ml concentration. Although the exact mechanism of this activity is not yet understood, it may be due to effect of surface area/volume ratio of the cells, which results in an enlargement of membrane or cells shrinkage and membrane proteins interaction (Shinde et al., 1999).
Figure 4. Membrane stabilization activity of Tulsi honey (A), Saffron honey (B), Ajwain honey (C) and Chambal honey (D) at different concentrations: (Mean ± S.D for n=3)
Figure 5. Comparative study for anti-inflammatory activity of different honey samples using HRBC membrane stabilization method. (Mean ± S.D for n=3)
Honey exhibit different multiple biological activities like anti-inflammatory, antioxidant, antibacterial etc. due to presence of different phytochemical compounds. Aim of this study was to investigate physicochemical characteristics and membrane stabilization effect of different honey samples to confirm its nutritional and therapeutic quality. All evaluated honey samples were acidic and were within the limits of standards prescribed. Longer shelf life during storage can be achieved as moisture content of all honey samples are within the standard range (<20%). Results of Ash content of different honey samples was in acceptable range. Membrane stabilization study revealed that as concentration of honey increases the membrane stabilization activity also increases which is indicative of their anti-inflammatory activity. Finally the present investigation concludes that, physicochemical properties, anti-inflammatory activity and quality of honey varied depending on the botanical origins, storage conditions and handling.
The authors are thankful to the SCES’s, the management of Indira College of pharmacy, Pune for providing necessary infrastructure and facility to conduct this research work.
Conflict of interest
Authors explicitly declare that there is no conflict of interest.
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