A New fatty acid and some triterpenoids from propolis of Nkambe (North-West Region, Cameroon) and evaluation of the antiradical scavenging activity of their extracts

Abstract The aim of this work was to evaluate in vitro antiradical scavenging activity of propolis from Nkambe (North-West, Cameroon). The polyphenol content of the acetone extract was evaluated using the Folin-Ciocalteu reagent as 0.166±0.008 gGAE/100 gRM. Antiradical scavenging activity of hexane and acetone extracts was carried out on DPPH using ascorbic acid as standard. The results showed that the extracts possess antiradical activity with IC50 of 141 μg/mL and 267 μg/mL for acetone and hexane extracts, respectively. The column chromatography separation on silica gel of the hexane fractionyielded compounds 1 to 3. The structures of these compounds were elucidated by NMR and mass spectrometry data as Lupenone (1), a mixture of α and ß-Amyrin (2) and lastly Hexatriacontanoic acid (3) which was described for the first time from propolis.


Introduction
Propolis is a resinous, sticky and balsamic substance of viscous consistency, produced by bees principally collected from resin of buds and exudates of plants and their secretions [1]. This substance has important pharmacological activities such as antiplasmodial [2], analgesic [3], antimicrobial [4,5], vasodilatory [1], antiinflammatory [6], antifugal [7], antioxidant [8], anti-ulcer [9], ostrogenic [10], antiviral, antiprotozoal, antiparastic, antitumor, hepatoprotective and cardioprotective properties [11]. In Cameroun, propolis is used locally to treat illnesses such as dysentery, stomachache, asthma, infertility, ulcers, tooth aches, fever, burns and different forms of inflammation [3,9]. Many studies on different samples show that propolis chemical composition is difficult to standardize because it is highly dependent on a number of factors such as local flora and environmental conditions of the site of collection of resin from plants for production of propolis. This is why propolis from areas not yet studied seems to be a promising source of new bioactive molecules [12]. From different botanical and geographical origins of the world, more than 300 compounds including volatile organic compounds, flavonoid aglycones, phenolic acids and their esters, phenolic aldehydes, alcohols and ketones, sesquiterpenes, quinones, coumarins, steroids, amino acids were reported to have been isolated from propolis [8,13]. Amongst these compounds, 241 of them were reported for the first time from the year 2000 to 2012 [14]. Despite the chemical variability of propolis, it always possesses promising and considerable biological activities [15].
This present study consisted of evaluating the in vitro antiradical activity of the hexane and acetone extracts of propolis from Nkambe and to purify the hexane extract in order to obtain pure compounds which will be identified based on their spectroscopic data ( 1 H NMR, 13 C NMR and MS).

Materials and Methods
The propolis sample under investigation was harvested during the month of April 2014 by a bee farmer in Njap village, Nkambe central subdivision, Donga-Mantung division, the North-West Region of Cameroon.

Extraction and isolation
To extract secondary metabolites, 1 kg of propolis was extracted with 5 L of acetone by maceration with intermittent stirring at intervals of 3 hours during 72 hours after which it was filtered on a N°1 Whatman filter paper and evaporated using a rotary evaporator to near dryness to obtain a crude acetone extract. This process was repeated three times in order to optimize the extraction process. The acetone extract obtained was partitioned using liquid-liquid extraction with hexane to obtain the hexane extract. 60 g of this hexane extract was subjected to column chromatography on 360 g of silica gel with the gradient eluting system hexane (Hex)-dichloromethane (CH 2 Cl 2 ) (100:0 → 0:100) followed by CH 2 Cl 2 -methanol (MeOH) (100:0 → 70:30). Fractions of 100 mL where collected regularly and concentrated on a rotavapor. This process yielded three compounds: Lupenone (1, 200 mg) [16], a mixture of α-amyrin (2a) and β-amyrin (2b) (85 mg) [17] and hexatriacontanoic acid (3, 25 mg).

Total polyphenol content
This was done according to the method described elsewhere [18]. A 100 μL of extract (200 μg/mL) were added to 200 μL of Folin-Ciocalteu followed by addition of 2000 μL of distilled water. The mixture was agitated for 3 minutes. After this, 1000 μL Na 2 CO 3 (20%) was added to the mixture and incubated in a dark cupboard at room temperature for 1 hour. The absorbance of the resulting solution was then measured at 760 nm on a spectrophotometer, with a methanol solution used as negative control. The preparation of the positive control, gallic acid (200 μg/mL) was subjected to the same treatment as the test sample. The results were expressed in term of gram equivalent of gallic acid per 100 g of raw matter (gGAE/100 gRM). The quantification of polyphenolic compounds was done with respect to a linear standardization curve obtained at different concentrations (20 to 120 μg/mL) of gallic acid in the form y=2.428x+0.033 whose correlation coefficient R was 0.994.

Evaluation of DPPH antiradical scavenging activity
The DPPH (1,1-diphenyl-2-picrylhydrazyl) antiradical scavenging activity of the extracts was done according to the method described elsewhere [12] with slight modifications. To 2 mL of the solution of the test sample prepared at different concentrations, 1 mL of a methanol solution of DPPH (100 μg/mL) was added. The mixture obtained was then stored at room temperature for 1 hour after which the absorbance was then measured at 517 nm against a negative control (6 mL of MeOH and 1 mL of DPPH solution) on a spectrophotometer. The positive control was ascorbic acid. A mother solution of sample at the concentration of 120 μg/mL was prepared by dissolving 5000 μg of extract 41.6 mL of MeOH. From this, five other solution concentrations were obtained from the mother solution making a total of six solutions at six concentrations: 120, 100, 80, 60, 40 and 20 μg/mL. The test at each concentration was done in triplicates, the absorbance was mesured and the percentage inhibition calculated according to the following equation [12].
Where A t0 = absorbance of negative control (without any anti-oxidant substance); A t1 = absorbance of tested samples.
Ethical approval: The conducted research is not related to either human or animal use.

Isolation and structural elucidation of compounds
The Hexane fraction of propolis of Nkambe was separated by column chromatography of silica gel yielded four compounds (1-3) among which one new compound (3). The structures of known compounds Lupenone (1), α-Amyrin (2a), and β-Amyrin (2b) were elucidated by comparison of their spectral data with those described in the literature [16,17].
Compound 3 was obtained has a white powder from the Hex/CH 2 Cl 2 9:1 fraction. Its molecular formula was established as C 36 H 72 O 2 by TOF-MS-ESI+ analysis which showed a quasi-molecular ion peak at m/z 537.3 [M+H] + . In its 1 H NMR spectrum a signal of three protons triplet at δ H 0.80 (3H, t, J = 6.0 Hz) indicates the presence of a terminal methyl group. A signal at δ H 1.20-1.32 (6H, brs, -(CH 2 ) 32 -) indicated the presence of a straight chain of 32 carbon atoms. The spectrum displayed a signal at δ H 2.30 (2H, t, -CH 2 -COOH) for methylene protons attached to a carboxylic group. On the basis of the NMR spectra it was inferred that compound 3 is an aliphatic acid. The 13 C NMR spectrum of compound 3 confirmed this suggestion by exhibiting important signals for carboxylic carbon at δ C 180.1 and methyl carbon at δ C 14.3 (C-36). The remaining methylene carbon resonated between δ C 34.1-29.0. Mass spectral studies offered further support to the above assignment. The molecular ion peak at m/z 537.3 gave the molecular formula C 36 H 72 O 2 . The peak at m/z 532.3 was due to the loss of two H 2 from molecular ion. The loss of an C 9 H 19 radical from the parent ion gave an ion which appeared at m/z 409.2 and this ion underwent successive loss of C 13 H 27 • and C 3 H 7 • units to give ions appearing at m/z 227.1 and 185.0 successively (Figure 2). The isolated aliphatic acid was therefore identified as Hexatriacontanoic acid.

Total polyphenol content and antiradical activity
The total polyphenol content was performed only on the acetone extract since phytochemical screening indicated that no phenolic compound was present in hexane extract. These results indicated the presence of polyphenolic compounds in the ethyl acetate extract and the total phenolic content of the said acetone extract was found to be 0.166±0.008 gGAE/100gRM. This value is less that that obtained by Talla and co-workers for propolis of Ngaoundal 2.32 ± 0.37-8.64 ± 0.47 gGAE/100gRM [18] and also those of Njintang and co-workers 10.99 ± 2.56-12.12 ± 2.24 g/100g [19] for some Cameroonian samples. This difference could be explained the difference in environmental conditions and local flora of site of collection of the propolis samples.
According to Melo and co-workers [20], anti-oxidant activity can be classified based on the performance of crude extracts as follows: good activity (IC 50 < 69 μg/ mL); moderate activity (69 μg/mL < IC 50 < 161 μg/mL); low activity (IC 50 > 161 μg/mL) [20]. The percentage inhibition was dose-dependent or concentration-dependent. The IC 50 were deduced by graphical means and presented in table 1 and higher values imply low activity and vice versa. The acetone extract had a higher activity with IC 50 of 141 μg/mL compared to hexane extract with IC 50 267 μg/mL. This could be explained by the absence of polyphenols in the hexane extract because the higher activity of acetone extract corroborates with its polyphenol content 0.166 ± 0.008 gGAE/100gRM. Ascorbic acid (vitamin C) possesses higher anti-radical activity (IC 50 = 9 μg/mL) than the tested samples.

Conclusion
Evaluation of antiradical activity carried out on the acetone extract showed a moderate one with percentage inhibition of 50.37% with an IC 50 of 140 μg/mL while that of the hexane hexane extract showed low antiradical activity with percentage inhibition of 38.75% and IC 50 of 267 μg/ mL. The acetone extract has low polyphenols content of 0.166 ± 0.008 gGAE/100gRM. Column chromatographic separation on silica gel of the hexane extract yielded compounds 1 to 3. The structures of these compounds were elucidated based on NMR and mass spectroscopic data   lup-20(29)-en-3-one (1), a mixture of α and ß-amyrin (2) and lastly hexatriacontanoic acid (3) which is described for the first time from propolis.