Physical-Chemical Characterization and Thin Layer Chromatographic Comparison of a Local Propolis with the Mangifera indica Resin

Mango (Mangifera indica) is an abundant tree of Cuban flora that is known for many healing properties and a deposit of bees as a source of raw materials for the propolis production. This work aimed to determine if the resin of mango felled bark was used by honeybees in the elaboration of a specific type of propolis. This could be possible by qualitatively comparison of its samples with the mango resin and the quality of this propolis through its physicochemical characteristics. To do this, propolis was collected and hydroalcoholic extracts were prepared. From this, botanical origin was determined by the technique of thin layer chromatography. Humidity, oxidation index, wax, resin and mechanical mixtures were evaluated from raw material. Results demonstrated that the samples of propolis collected presented a chromatographic profile similar to mango resin. In the samples of propolis, it was determined that quercetin and mangiferin are present in their qualitative composition of secondary metabolites. On the other hand, the mango propolis satisfies the requirements established by international regulations for good quality propolis in terms of humidity, oxidation index and mechanical mixtures.


Introduction
Propolis is a resinous material of varied coloration that has been studied extensively in recent years. It is elaborated by honeybees for different purposes including the protection of the hive against   10,11,12 and its medical and pharmacological properties.

Pharmaceutical and Biosciences Journal
A comparative study of mango bark resin with propolis of the same origin has not been analyzed until now. In the experimental apiary of the Beekeeping Research Center (CIAPI for its acronym in Spanish), which is located in a plantation with a very significant representation of Mangifera indica, we have observed honeybee collecting resin from felled mango trees. Samples of this resin and propolis from the hives of the same apiary were taken. The aim was to determine if the resin of the mango felled bark was used by the bees for the elaboration of a specific type of propolis. This could be possible by the qualitative comparison of its extracts with the mango resin and the quality of this propolis through its physical-chemical characteristics.

Obtaining propolis and mango resin
In the month of March, after four weeks of cleaning the hives that belong to the study apiary of the Beekeeping Research Center in Cuba (23° 1´43.80´´N of latitude and 82° 27´28.61´´O of longitude), resin samples were taken by scraping the felled barks of Mangifera indica trees. These are arranged no more than 20 meters from the study hives. Propolis collection was carried out at the same time using the same method, scraping, and samples only were taken on the tops or roof of the study hives.

Samples preparation
Three samples of resin and 12 of propolis were taken to the laboratory where they were placed in a freezer overnight and subsequently macerated. This macerates were put in vials and 99% alcohol added to a 1g / 5ml ratio. They were left in extraction at room temperature, protected from light and with occasional agitation for 24 hours, then the solution was filtered and the extract saved until used. These samples were called mango resin and mango propolis.

Reference substances
Nemorosone, quercetin and mangiferin standards were from Shaanxi Sciphar Hi-Tech Industry and Sigma-Aldrich. They were diluted in ethanol with an approximately 99, 95 and 90% of purity was used respectively.

Thin Layer Chromatografhy (TLC)
Samples of mango resin, mango propolis and resin of mango (from procedure to determine resin in propolis in the section of Physicochemical characterization) were run in a TLC.
A glass chromatographic camera was used and worked at room temperature (25-29° C). Silica gel plates on glass support were used for qualitative analyzes. The application was made with the use of capillaries with spontaneous drying of the applications. An upward runs was developed using three solvent systems as mobile phases: mixture hexane: ethyl acetate (3:2), another ethyl acetate: acetic acid: formic acid: water (7:1:1:1) and hexane: ethyl acetate (7:3).
Solvents hexane: ethyl acetate (7:3) and toluene: ethyl acetate: formic acid (6:5:1) were used for the two-dimensional TLC. The application was made one centimeter from the bottom edge and the run was approximately eight centimeters. The distance between each application was one centimeter. Result of all run profile of the samples was visualized through an UV lamp at 254 nm. The exposure of the plates to iodine vapors was used as developer.

Physicochemical characterization
All analyzes were done following the methodology proposed by the Norma Argentina IRAM-INTA 15935-1 13 . The tests were performed in triplicate and the results were expressed in percentages, except for the oxidation index that was expressed in seconds.
Humidity was determined using the thermogravimetric method.
One gram of the raw propolis was weighed in a previously tared capsule and placed in an oven at 105° C; subsequently, it was allowed to cool in a desiccator and the process was repeated until a constant weight was obtained (two successive weights should not differ by more than ± 5 mg).
The determination of extractable substances (wax) in n-hexane was carried out by weighing two grams of sample arranged in a cellulose cartridge; condensation was carried out using the soxhlet technique from a volume of 200 ml of n-hexane for six hours, then the volume left in the ball was measured, 10 ml were taken, which were dried in an oven at 105°C and weight of dried wax was used to estimate its proportion in the total sample. The procedure to determine resin was very similar. The remainder of each sample resulting from the extraction of extractable substances with hexane was placed in the body of a soxhlet extractor. A new ethanol extraction was performed. At the end, the ethanol content was brought to a volume of 100 ml. An aliquot was taken that was placed on plates and dried at 80° C and weight of dried wax was used to estimate its proportion in the total sample. sulfuric acid solution and stirred for one minute. Finally, a drop of 0.1 N potassium permanganate was added and the chronometer was started while stirring constantly. The time, in seconds that the solution bring to discolor was recorded.

Thin Layer Chromatography (TLC)
The TLC is a simple, economical and easiest available technique, widely employed in plants analysis. In our study, the TLC has been employed to qualitatively analyze the origin source of collected propolis from CIAPI´s apiary and the presence of some of its secondary metabolites.
Although only three compounds were evaluated as standards  In the chromatographic runs with mixture hexane: ethyl acetate (7:3), a less polar system, samples of mango propolis, mango resin and resin of propolis were applied together with the reference substance, nemorosone (Fig 2). They showed a significant diversity of separate compounds corresponding to different secondary metabolites. The three samples applied exhibited a chromatographic profile with seven coincident bands of equal Rf. Regarding the references substances employed, well-defined bands with equal Rf to nemorosone were observed in the three samples.
The development of the bi-dimensional TLC with the third solvent system is showed in figure 3. For this case, only the mango propolis was run. A first run was made with hexane: ethyl acetate (7:3) while in a second system was used the mixture toluene: ethyl acetate: formic acid (6:5:1).

1). A) λ = 254 nm. B) developed with iodine vapors
The profile showed that the band appeared in the previous chromatographies with the same Rf as nemorosone standard does not correspond to this compound. In this case, when resolving the bands in two dimensions it was observed that is not the prenylated compound that appears in the bands identified with the nemorosone.

Samples of mango propolis, mango resin and resin of propolis
were applied together with the reference substance mangiferin for runs with ethyl acetate: acetic acid: formic acid: water (7:1:1:1) (Fig 4).   From this information it can be concluded that values for humidity, oxidation index and mechanical mixture were suitable for a propolis of good quality according to the international regulations consulted. The wax showed a mean above the highest value proposed by the same regulation whiles the resin under the lowest values. In both cases, despite the fact that values do not comply with the regulation, is notable to observer that even then the means are very close to the limits.  In the plates, the bands corresponding to the nemorosone were identified in mango propolis and resin of propolis with good resolution (Fig 1 and 2 Taking into account these antecedents and to be sure that the bands found in chromatographies were actually those corresponding to the nemorosone standard used or some other compound derived from it, a two-dimensional TLC was performed in which the second run was used a solvent system that according to reports of the literature solves the flavonoid bands well 14 .
The results observed in figure 3  The waxes, which are associated with a low quality of tint 24 , were above the maximum limits established by the international standards consulted. While resin, the main quality parameter of raw propolis, obtained an average below the minimum limits of the same standards. In this fraction some authors said that are the compounds to which the biological activity is attributed. The higher the value of this fraction, the better it will be in terms of performance 24 .
Mechanical mixtures, a visible physical characteristic showed an average below the recommended maximum limits.

Conclusions
It can be concluded that the propolis collected from the CIAPI apiary originates from the mango resin. The Mangifera indica resin and mango propolis studied qualitatively present a similar composition. Finally, mango propolis meets the requirements established by international regulations for propolis of good quality in terms of humidity, oxidation index and mechanical mixture.

Conflicts of interest
The authors declare that no conflicts of interest regarding this paper exist.