Screening and Bioguided Fractionation of Mimosa pigra L. Bee Pollen with Antioxidant and Anti-Tyrosinase Activities

The bee pollen used in this study was collected from A. mellifera that had access to mimosa (Mimosa pigra L.) flowers in the Chiangmai province, Thailand in 2017. The pollen was stored at room temperature until further analysis. A small amount of dry bee pollen was investigated with a scanning electron microscope (SEM). The morphology and characteristics of the bee pollen was recorded under SEM at 5,000X magnification. Our findings were compared to the identification results described for that plant species by Caccavari (2002) and El Ghazali et al. (1997). The sample was then prepared according to the procedure as described in details in the following subsections. The overall extraction and enrichment procedure are shown in Fig. 1.

Fig. 1

The bee pollen (140 g) was mixed with 800 mL of methanol (MeOH), shaken at 100 rpm, 15°C for 18 h, and then centrifuged at 5500 rpm, 4°C for 15 min. The supernatant was collected while the solid residue (pellet) was re-extracted three more times in the same manner with 800 mL of MeOH each time. The four MeOH extracts were combined together and the solvent evaporated under reduced pressure at a maximum temperature of 40–45°C to yield the crude MeOH extract of mimosa flower bee pollen (CME). The CME was kept at −20°C in the dark until used.

The CME was sequentially partitioned by MeOH (high polarity), dichloromethane (DCM; medium polarity) and hexane (low polarity). To this end, the CME was dissolved in MeOH until it was not sticky and then mixed with an equal volume of hexane in a separating funnel and left to phase separate, whereupon the upper hexane phase was collected. The lower MeOH phase was then further extracted with hexane in the same manner twice more, and the hexane extracts were pooled and evaporated under reduced pressure at a maximum temperature of 40–45°C to yield the hexane partitioned extract of mimosa flower bee pollen (HXMBP). Meanwhile, the residual MeOH phase was then extracted with an equal volume of DCM three times in the same manner as above (except the DCM phase was the lower layer), with the pooled DCM extracts evaporated as above to yield the DCM partitioned extract of mimosa flower bee pollen (DCMMBP). Finally, the residual MeOH phase was evaporated as above to yield the MeOH partitioned extract of mimosa flower bee pollen (MTMBP). All three partitioned extracts were tested for their antioxidant and anti-tyrosinase activities as detailed below.

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, modified from Chantarudee et al. (2012), was used to assay the antioxidant activity. Five different concentrations of each sample were prepared in dimethyl sulfoxide (DMSO). For each concentration, 20 μL of the sample was mixed with 80 μL of 0.15 mM DPPH in MeOH and incubated at room temperature for 30 min. The absorbance was measured at 517 nm (A₅₁₇) using a microplate reader. Ascorbic acid (vitamin C) was used as the standard reference. The antioxidant activity and the effective concentration at 50% (EC₅₀) were then calculated as mentioned in Chantarudee et al. (2012).

The anti-tyrosinase activity was determined as reported by Zhang et al. (2015) with minor modifications. Five different concentrations of the sample in DMSO were prepared. The reaction mixture contained 120 μL of 2.5 mM L-3,4-dihydroxyphenylalanine (L-DOPA) in a phosphate buffer, 30 μL of 80 mM phosphate buffer (pH 6.8) and 10 μL of the sample solution in DMSO. The mixtures were mixed and then pre-incubated at 25°C for 10 min before 40 μL of mushroom tyrosinase (165 units/mL) in phosphate buffer was added and incubated at 25°C for 5 min. The absorbance of the sample was measured at 475 nm (A₄₇₅) using a microplate reader. Kojic acid was used as the positive diphenolase inhibitor standard. Each sample was performed and measured in triplicate. The tyrosinase inhibition (%) and the inhibitory concentration at 50% (IC₅₀) were calculated as mentioned in Zhang et al. (2015).

The extract with the highest antioxidant activity (DCMMBP) was selected for fractionation by SiG60-CC. The 500-mL column was packed with fine SiG60 (Merck, for column chromatography). The DCMMBP partitioned extract (7.24 g) was dissolved in 40 mL of MeOH and combined with 20 g of rough SiG60 (Merck for CC). When dry, it was poured over the surface of the packed SiG60 column and then firstly eluted with 500 mL of DCM, followed by 2,000 mL of 10:1 (v/v) DCM: MeOH and then 1,000 mL of MeOH, respectively. Eluted fractions (10 mL each) were collected, and the solvent was removed by evaporation under reduced pressure at a maximum temperature of 40–45°C. The pattern of chemical compounds in each fraction was profiled by thin layer chromatography (TLC; see below). Fractions with the same TLC pattern were assumed to be chemically similar and were pooled. Each fraction, or pooled fraction, was then tested for its antioxidant activity using the DPPH assay as above.

Sephadex LH-20 gel was immersed in absolute MeOH overnight to allow the beads to swell. Cotton was placed at the bottom of a 250-mL column and the swollen Sephadex LH-20 gel was packed into the column to 4/5 of the column height. The active fraction was dissolved in absolute MeOH until it was not too viscous and then loaded carefully onto the top of the Sephadex LH-20 gel using a Pasteur pipette, with the packed column valve open to allow the partitioned extract to be absorbed into the gel. The column was then eluted with 500 mL of absolute MeOH, collecting 5 mL fractions. A small amount of each fraction was resolved by TLC (see above) and fractions with a similar TLC profile were pooled. All fractions or pooled fractions were then evaluated for their antioxidant activity using the DPPH assay as above.

A 5 × 5 cm² TLC plate with silica as the immobile phase was prepared. The test sample was spotted onto the solvent front line of the plate by a capillary tube, allowed to dry at room temperature, and then resolved in one direction using the appropriate mobile phase solvent of (a) 10:1 (v/v) DCM: MeOH or (b) 10:0.5:1 (v/v/v) DCM: ethyl acetate (EtOAc): MeOH. The resolved compounds on the TLC plate were visualized under UV light at 254 nm or by dipping in 3% (v/v) anisaldehyde in MeOH and heating over a hot plate . The identification of the yellow solid compound in fraction DCMMBP3-2, named compound I, was done based on the chemical structure analysis (NMR) and based on the comparison to the flavonoid isolated from the aerial parts of Cyclotrichium origanifolium (Guzel et al., 2017).

Fig. 2

Fig. 3

Each enriched fraction was evaporated and analysed. Briefly, the evaporated sample was dissolved in an appropriate deuterated solvent (MeOH-D4, Merck) at a ratio of 5-20 mg of compound to 600 μL of deuterated solvent. It was then transferred to an NMR tube and shaken until well combined. The NMR spectrum was recorded by a Varian Mercury plus 400 machine operated at 400 MHz for ¹H-NMR and a Bruker AVANCEIII 400 MHz machine for ¹³C-NMR nuclei in order to detect the functional groups using tetramethylsilane as the internal standard. The chemical shift in δ (ppm) was assigned with reference to the signal from the residual protons in the deuterated solvents, while the chemical shift and J coupling value were determined using the MestReNova version 12.0.3 software.

Experiments were performed in triplicate. Numerical data are reported as the mean ± standard deviation (±SD), determined in the Microsoft Excel 2016 software.

Under SEM, the morphology of the mimosa (Mimosa pigra L.) flower bee pollen was observed to be from oblate to spherical in shape with four (tetrad) pollen subunits (Fig. 2).

The characteristics (weight, yield and appearance) for crude (CME) and partitioned extracts (MTMBP, DCMMBP, HXMBP) are presented in the Tab. 1. The results for the antioxidant and anti-tyrosinase activity of these bee pollen extracts, are also introduced in the Tab. 1.

For their antioxidant activity, the obtained EC₅₀ values are shown in comparison to that for ascorbic acid as the standard reference. Only the DCMMBP extract was the most active (EC₅₀ of 192.1 μg/mL), although this was still an over two-fold higher EC₅₀ value (so less effective) than ascorbic acid (EC₅₀ of 89.8 μg/mL).

The three partitioned extracts were also tested for their tyrosinase inhibitory activity, with the obtained IC₅₀ values shown in Tab. 1 in comparison with that for kojic acid as the standard reference. None of the crude extracts showed any notable anti-tyrosinase activity, with IC₅₀ values of >500 μg/mL compared to 8.58 μg/mL for kojic acid.

Since the DCMMBP provided the best antioxidant activity (192.1 μg/mL), it was further fractionated by SiG60-CC. From 7.24 g of DCMMBP a total of ninety-six fractions were collected. After a comparison of the TLC profiles and pooling fractions with a similar pattern, three different fractions (DCMMBP1-3) were obtained. Their weight, yield and appearance are summarized in Tab. 2. These three pooled fractions were then tested for their antioxidant activity, with the derived EC₅₀ values reported in Tab. 2. Fraction DCMMBP3 gave the highest antioxidant activity (EC₅₀ of 112.2 μg/mL) which was only around 1.4-fold higher numerically from that for ascorbic acid (EC₅₀ of 81.1 μg/mL).

In addition, fractions DCMMBP1-3 at 50 μg/mL were tested for their anti-tyrosinase activity, with the obtained results shown in Tab. 2. All three fractions had a very low anti-tyrosinase activity with essentially no activity (at 50 μg/mL) for DCMMBP3.

Since fraction number DCMMBP3 provided the best antioxidant activity (EC₅₀ of 112.2 μg/mL), it was further enriched by Sephadex LH-20 size exclusion chromatography to yield a total of sixty-six fractions. After a pooling of fractions with similar TLC profiles, two different fractions (DCMMBP3-1 and DCMMBP3-2) were obtained. Their weight, yield and appearance are summarized in Tab. 2. With respect to their antioxidant activity, fraction DCMMBP3-1 had the highest antioxidant activity (EC₅₀ of 121.3 μg/mL), while DCMMBP3-2 had essentially no antioxidant activity. The antioxidant activity for DCMMBP3-1 (EC₅₀ of 121.3 μg/mL) was only slightly less powerful than its parental fraction (DCMMBP3; EC₅₀ of 112.2 μg/mL) although DCMMBP3-1 accounted for only about 38% of the antioxidant activity in DCMMBP3 (after taking account of the 58.2% yield loss from SiG60-CC of DCMMBP and the 12.6% yield loss from Sephadex-LH20-CC of DCMMBP-3). It is unclear if this apparent activity loss represents the removal of synergistic compounds or cofactors into DCMMBP3-2 or is simply a yield loss in the CC.

After Sephadex LH-20 size exclusion CC, the chemical composition of the two obtained fractions was tested by TLC (Fig. 3). The smear on the TLC plate indicated that fraction DCMMBP3-1 seemed to contain more than one chemical component of a similar polarity and molecular weight, and so fraction DCMMBP3-1 was reported as a mixture. In contrast, only a sharp band was observed on the TLC plate for fraction DCMMBP3-2, which indicated it was potentially a pure compound. Therefore, the chemical structure of the compound, named compound I, in fraction DCMMBP3-2 was analyzed by NMR.

Compared to the flavonoid isolated from the aerial parts of Cyclotrichium origanifolium (Guzel et al., 2017), the obtained NMR peaks in the chemical shift pattern were ¹H-NMR (400 MHz, MeOH-d₄) δ: 7.30 (d, J=8.5 Hz, 2H), 6.81 (d, 8.5 Hz, 2H), 5.88 (q, J=2.2 Hz, 2H), 5.31 (dd, J=13.0, 3.0 Hz, 1H), 3.09 (dd, J=17.1, 13.0 Hz, 1H) and 2.67 (dd, J=17.1, 3.0 Hz, 1H). ¹³C-NMR (100 MHz, MeOH-d₄) δ: 197.8, 168.4, 164.9, 159.0, 131.1, 129.0, 116.4, 103.4, 97.1, 96.3, 80.5 and 44.0 (Supplement 1). The compound I was identified as naringenin (Fig. 4).

Fig. 4

When the mixed fraction DCMMBP3-1 was analyzed for its chemical structure by NMR the obtained NMR peaks of chemical shift pattern were ¹H-NMR (400 MHz, MeOH-d₄) δ: 7.56-7.41 (m, 3H), 7.39 (dd, J=9.2, 5.5 Hz, 1H), 7.13-7.05 (m, 2H), 7.05-7.02 (m, 1H), 7.02-6.91 (m, 4H), 6.91-6.65 (m, 7H), 6.40 (ddt, J=20.6, 15.7, 5.5 Hz, 3H), 4.64 (s, 1H), 3.86-3.71 (m, 14H), 3.52 (s, 2H), 3.46 (dd, J=18.5, 7.7 Hz, 3H), 3.34 (s, 13H), 1.87 (dt, J=26.9, 7.1 Hz, 3H), 1.67 (s, 1H) and 1.66-1.51 (m, 4H). ¹³C-NMR (100 MHz, MeOH-d₄) δ: 169.2, 149.8, 149.5, 149.3, 144.7, 142.4, 142.1, 128.6, 128.3, 123.5, 123.3, 121.3, 116.6, 116.3, 115.4, 112.3, 111.7, 106.9, 106.5, 56.9, 56.6, 56.5, 49.9, 49.7, 49.5, 49.3, 49.1, 48.9, 48.6, 48.4, 47.6, 47.1, 45.7, 40.2, 39.9, 38.3, 38.1, 28.9, 27.9 and 27.7 (Supplement 2). Considering the ¹H- and ¹³C-NMR, a phenol derivative was suggested to be the major compound.