Chemical constituents of Brazilian Propolis from the state of Bahia and their growth inhibitory activities against cancer cells

Abstract

A novel 2-phenoxychromone (1) and five known flavones (2-6) were isolated from northeastern Brazilian propolis in the state of Bahia. The chemical structures of these six compounds were determined by spectroscopic investigations and single-crystal X-ray analysis. The isolated compounds showed growth-inhibitory activities, in varying degrees, against human tumor cell lines. This is the first report on the discovery of a novel 2-phenoxychromone from propolis.

A novel 2-phenoxychromone (1) and five known flavones (2–6) were isolated from Brazilian propolis in the state of Bahia.

Keywords:

• Brazilian propolis
• Bahia state
• 2-Phenoxychromone
• anti-cancer

Propolis is a solid substance that consists of resin, wax and several active ingredients. This substance is made by honeybees (Apis melifera) from several plant exudates. The main role of propolis is not only to protect beehives from invaders, rain and wind but also to maintain hygiene. Propolis has been used in traditional folk medicine. There are many reports on the biological activities of propolis, including its anti-bacterial [1–3], anti-viral [2–4], anti-inflammatory [5] and anti-cancer properties [6–12]. However, the chemical composition of propolis differs depending on the habitat of the bees and plant sources.

In the course of investigating the constituents of several varieties of propolis, we have discovered a novel diterpenoid unique to brown Brazilian propolis from Parana which has potent anti-proliferative activity [13] in several cancer cell lines. In this study, we investigated northeastern Brazilian propolis from the Atlantic forest of Bahia, commercially called “Special propolis” (Nordeste Co., Ltd., Tokyo). Brazilian propolis has been grouped into 12 chemical types according to their botanical origin [14], and this Brazilian propolis from Bahia state (BA propolis) is thought to be categorized as type 7. However, there are limited reports on the chemical components found in BA propolis and their bioactivities. In preliminary studies, we found that the ethanol extract of BA propolis has an anti-proliferative effect on cancer cells as well as anti-bacterial activity and is slightly more potent than ethanol extracts of Brazilian green propolis, which is a popular propolis in many countries. We also found that BA propolis does not include artepilin C, which is a major compound found in Brazilian green propolis [15,16]. In this study, we describe the identification of the key compounds isolated from ethanol extracts of BA propolis.

Materials and methods

Extraction and isolation

Crude of BA propolis were distributed in the Atlantic forest region of Bahia state, northeastern Brazil and is collected by Nordeste Co., Ltd. BA propolis was extracted with 95% ethanol at room temperature for a day. This extraction process was repeated twice (three times in total). Each filtrate were concentrated under reduced pressure and combined to give a crude residue (1.92 g), which was chromatographed in a silica gel column, using a stepwise gradient of

CHCl₃-Methanol = 100:0–1:1 to yield ten fractions (Fr. 1-10) according to their TLC profiles. Fr. 10 (140.79 mg) was purified by preparative HPLC using by liner gradient mixture of Acetonitrile-H₂O = 40:60–100:0 (60 min.), to yield compound 1 (19.8 mg) and 2 (16.1 mg). Fr. 6 (277.25 mg) was fractionated by preparative HPLC using liner gradient mixture of Acetonitrile-H₂O = 40:60–100:0 (60 min.) to give compound 3 (6.7 mg). Fr. 7 (423.16 mg) was separated by preparative HPLC elution with liner gradient mixture of Acetonitrile-H₂O = 40:60–100:0 (60 min.) to give four fractions (Fr. 7a-7d). Further purification of Fr. 7b (63.3 mg) was accomplished by preparative HPLC using liner gradient mixture of Acetonitrile-H₂O = 30:70–60:40 (60 min.) to yield five fractions (Fr. 7b1-7b5). Among these fractions, Fr. 7b3 and Fr. 7b5 were purified compound 4 (7.2 mg) and 5 (23.0 mg), respectively. Fr. 7b1 (10.8 mg) was achieved with preparative HPLC using liner gradient mixture of Acetonitrile-H₂O = 30:70–60:40 (60 min.) to yield compound 6 (5.9 mg).

Compound 1 (5,7-dihydroxy-2-(3,4-dihydroxyphenoxy)-4H-chromen-4-one) was isolated a pale yellow crystal (m.p. 137 °C); IR (KBr) cm⁻¹: 3321, 1658, 1613, 1503, 1427, 1357, 1219, 1142, UV λ_max (MeOH) nm (log ε) : 231.5 (4.09), 289.0 (3.83), ¹H-NMR (300 MHz, CD₃OD): δ_H 6.19 (1H, d, J = 1.8 Hz; H-6), 6.30 (1H, d, J = 1.8 Hz; H-8), 6.58 (1H, dd, J = 8.4, 2.7 Hz; H-6′), 6.68 (1H, d, J = 2.7 Hz), 6.84 (1H, d, J = 8.4 Hz; H-5′), 5.14 (1H, s), ¹³C-NMR (75 MHz, CD₃OD): δ_C 86.6 (C-3), 93.8 (C-8), 99.3 (C-6), 102.3 (C-10), 108.0 (C-2′), 111.3 (C-6′), 115.5 (C-5′), 144.0 (C-1′), 144.5 (C-4′), 146.6 (C-3′), 155.8 (C-9), 161.9 (C-5), 164.5 (C-7), 169.5 (C-2), 184.3 (C-4), and HR-ESI-MS: m/z 303.0509 [M+H]⁺ (calcd. for C₁₅H₁₁O₇; 303.0505).

Compound 2 (3-O-methylquercetin) was obtained as a pale yellow powder; ¹H-NMR (300 MHz, CD₃OD): δ_H 3.78 (3H, s; 3-OCH₃), 6.19 (1H, d, J = 1.8 Hz; H-6), 6.38 (1H, d, J = 1.8 Hz; H-8), 6.90 (1H, d, J = 8.4 Hz; H-5′), 7.52 (1H, dd, J = 8.4, 2.1 Hz; H-6′), 7.62 (1H, d, J = 2.1 Hz; H-2′) and ¹³C-NMR (75 MHz, CD₃OD): δ_C 60.6 (3-OCH₃), 93.5 (C-8), 98.6 (C-6), 104.7 (C-10), 115.2 (C-2′), 115.3 (C-3′), 121.1 (C-6′), 121.7 (C-1′), 138.4 (C-3), 146.6 (C-3′), 148.8 (C-4′), 156.8 (C-9), 157.2 (C-2), 161.9 (C-5), 164.7 (C-7), 178.8 (C-4).

Compound 3 (3,6,4′-trimethoxychrysin) was obtained as yellow powder; ¹H-NMR (300 MHz, DMSO-d₆): δ_H 3.75 (3H,s; 3-OCH₃), 3.79 (3H, s; 6-OCH₃), 3.86 (3H, s; 4′-OCH₃), 6.57 (1H, s; H-8), 7.14 (1H, d, J = 9.0 Hz; H-3′, 5′), 8.03 (1H, d, J = 9.3 Hz; H-2′,6′), 12.74 (1H,s; 5-OH) and ¹³C-NMR (75 MHz, DMSO-d₆): δ_C 55.4 (4′-OCH₃), 59.7 (3-OCH₃), 59.9 (6-OCH₃), 94.0 (C-8), 104.6 (C-10), 114.2 (C-3′, 5′), 122.1 (C-1′), 129.9 (C-2′, 6′), 131.1 (C-6), 137.5 (C-3), 151.5 (C-9), 152.3 (C-5), 155.2 (C-2), 157.4 (C-7), 161.3 (C-4′), 178.2 (C-4).

Compound 4 (3,6-dimethoxyapigenin) was obtained as a yellow oil; ¹H-NMR (300 MHz, Acetone-d₆): δ_H 3.87 (3H,s; 3-OCH₃), 3.87 (3H, s; 6-OCH₃), 6.58 (1H, s; H-8), 7.01 (2H, d, J = 9.0 Hz; H-3′), 8.03 (2H, d, J = 9.0 Hz; H-2′) and ¹³C-NMR (75 MHz, Acetone-d₆): δ_C 59.5 (3-OCH₃), 60.0 (6-OCH₃), 93.8 (C-8), 105.6 (C-10), 115.8 (C-3′, 5′), 122.0 (C-1′), 130.6 (C-2′, 6′), 131.2 (C-6), 138.1 (C-3), 152.4 (C-7), 153.0 (C-5), 156.3 (C-9), 157.1 (C-2), 160.3 (C-4′), 179.2 (C-4).

Compound 5 (6-methoxykaempferol) was obtained a pale yellow powder; ¹H-NMR (300 MHz, CD₃OD): δ_H 3.88 (3H, s; 6-OCH₃), 6.49 (1H, s; H-8), 6.90 (2H, d, J = 9.0 Hz), 8.08 (2H, d, J = 9.0 Hz) and ¹³C-NMR (75 MHz, CD₃OD): δ_C 59.8 (6-OCH₃), 93.6 (C-8), 103.7 (C-10), 115.1 (C-3′, 5′), 122.5 (C-1′), 129.5 (C-2′, 6′), 131.0 (C-6), 135.7 (C-3), 147.1 (C-2), 151.8 (C-9), 152.5 (C-5), 157.5 (C-7), 159.4 (C-4′), 176.4 (C-4).

Compound 6 (6-methoxyapigenin) was obtained as a pale yellow powder; ¹H-NMR (300 MHz, DMSO-d₆): δ_H 3.75 (3H, s; 6-OCH₃), 6.60 (1H, s, H-8), 6.78 (1H, s; H-3), 6.92 (2H, d, J = 8.2 Hz; H-3′), 7.92 (2H, d, J = 8.2 Hz), 13.08 (1H, s; 5-OH) and ¹³C-NMR (75 MHz, DMSO-d₆): δ_C 60.0 (6-OCH₃), 94.4 (C-8), 102.5 (C-3), 104.1 (C-10), 116.1 (C-3′, 5′), 121.3 (C-1′), 128.6 (C-2′, 6′), 131.5 (C-6), 152.5 (C-9), 152.9 (C-5), 157.5 (C-7), 161.3 (C-4′), 163.9 (C-2), 182.2 (C-4).

Cytotoxicity assays

In vitro cytotoxic activities of isolated compounds (1-6) were evaluated in DLD-1 (human colon cancer), MCF-7 (human breast cancer) and A549 (human lung cancer) cancer cell lines. The cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and were cultured at a density of 1 × 10⁴ cells or 5 × 10³ cells / 100 μL medium / well in 96-well microtiter plates at 37 °C in a 5% -CO₂ incubator. After 24 h of culture, the cells were treated with various concentrations of the propolis extract (4.7–28 μg/mL) or the isolated compounds (6.25–200 μM) for 24 h. The test agents were dissolved in DMSO solution, and were tested in triplicate. Growth inhibition in the cancer cells was evaluated by quantifying living cells using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). Briefly, after treatment with the test agents, the cells were incubated in medium containing 10% CCK-8 for 1 h at 37 °C in 5% CO₂, and the absorbance of formazan, which is produced by living cells from the chromogenic substrate tetrazolium salt (WST-8), was spectrophotometrically measured at 450 nm. The IC₅₀ values of the test agents were estimated by nonlinear regression analysis for growth inhibition of each cancer cell line using the GraphPad Prism program (Graphpad software Inc., La Jolla, CA).

Results and discussion

Compound 1 was obtained as pale yellow crystals (m.p. 137 °C), and its high-resolution (HR-) ESIMS exhibited a pseudo-molecular ion peak at m/z 303.0509 [M+H]⁺, corresponding to a molecular formula of C₁₅H₁₁O₇ (Δ + 0.4 mmu) and indicating eleven degrees of unsaturation. The IR spectrum showed absorption bands for a hydroxyl group (3321 cm⁻¹), an α, β-unsaturated carbonyl group (1658 cm⁻¹) and an aromatic sp² stretch (1613 cm⁻¹) [17]. The ¹H-NMR spectrum of compound 1 displayed signals from an olefinic proton (H-3), three protons typical of an ABX system at H-2′, 5′ and 6′ and a m-coupled aromatic doublet at H-6 and 8. The ¹³C-NMR spectrum showed fifteen carbon resonances, including two olefinic carbons (C-2, 3), twelve aromatic carbons (A- and B-ring) and an α, β-unsaturated carbonyl group (C-4) which was shifted upfield. These NMR spectroscopy data were very similar to those of a typical flavone.

The structure of compound 1 was elucidated primarily based on the heteronuclear multiple bond connectivity (HMBC) spectrum. Long-range ¹³C-¹H correlations (H-3/2, 4, 10, H-6/C-5, 7, 8, 10 and H-8/C-6, 10) indicated the presence of A- and C-rings (Figure 1). The A- and C-rings were also confirmed by the positive ion ESI-MS spectrum, which exhibited peaks at m/z 303 [M+H]⁺, 195 [M+H-108]⁺ and 153 [M+H-150]⁺. The fragmentation at m/z 153 resulted from a Retro Diels-Alder (RDA) cleavage of compound 1 at the C-ring. However, there were no HMBC correlations from a tri-substituted benzene ring (B-ring). Moreover, the chemical shift of C2 (169.5 ppm) and C1′ (144.0 ppm) were downfield to those of flavones (luteolin) [18]. These chemical shifts are close to capillarisin, a 2-phenoxychromone [19]. This evidence suggested that the B-ring and C-ring are linked by the ether bridge C1′-O-C2.

Figure 1. 2D-NMR correlations of compound 1 (bold line shows ¹H-¹H COSY correlation, solid arrows show HMBC correlations).

Furthermore, compound 1 formed a crystal structure suitable for single-crystal X-ray analysis. The connectivity of the B- and C-ring was confirmed by single-crystal X-ray analysis. The ORTEP drawing of the monohydrate of compound 1 is shown in Figure 2. Based on these results, it was revealed that compound 1 was identified as a 5, 7-dihydroxy-2-(3, 4-dihydroxyphenoxy)-4H-chromen-4-one, an unknown compound, and has the chemical structure shown in Figure 3. The crystallographic information files of compound 1 have been deposited in the Cambridge Crystallographic Data Centre (CCDC deposition number 1532831).

Figure 2. ORTEP drawing of the monohydrate of compound 1, as determined by X-ray crystal analysis.

Figure 3. Structures of compounds 1 - 6 isolated from the ethanol extract of BA Propolis.

The structures of known compounds 2 to 6 were determined by a combination of spectroscopic analysis and comparison with reported data. Compounds 2 [20], 32 [21,22], 4 [23], 5 [24] and 6 [25] were identified as 3-O-methylquercetin, 3, 6, 4′-trimethoxychrysin, 3, 6-dimethoxyapigenin, 6-methoxykaempferol and 6-methoxyapigenin, respectively. To the best of our knowledge, there are few reports that the compounds (2-6) were found in other types of propolis from geographically diverse areas. Especially, this is the first report describing the discovery of 2-phenoxychromone from propolis. For these reasons, the isolated compounds must be derived from plant sources but not honey bee metabolites. In addition, 2-phenoxychromones have mainly been isolated from Asteraceae (Artemisia [26], Achillea [27] etc.), Fabaceae (Piliostigma [28], Peltophprum [29] etc.), suggesting that they are possibly one of the plant sources of BA propolis.

The growth-inhibitory activities of the propolis extract and the isolated compounds (1-6) against the three human cancer cell lines DLD-1 (human colon cancer), MCF-7 (human breast cancer) and A549 (human lung cancer) were evaluated with the CCK-8 assay in vitro. As shown in Table 1, the extract of BA propolis showed obvious anti-proliferation activities against each the cancer cell line. Compounds 2, 3 and 4 also showed inhibition of cell growth with nearly equal activity in all the cell lines tested (Table 1). These compounds have a methoxy substituent at the 3-position in the chemical structure. There are several reports that 3-methoxy flavones inhibit cancer-cell growth [30–32]. These data suggest that the presence of the methoxy substituent at position 3 of the C-ring might play an important role in the growth inhibition of cancer cells by flavone compounds. Interestingly, it was demonstrated that compound 1, a novel 2-phenoxychromone derivative, has an anti-proliferative effect on cancer cells, even though its activity seems to be lower than that of the 3-methoxy flavones.

Table 1. The growth-inhibitory activity of constituents isolated from the ethanol extract of BA propolis against human cancer cell lines.

Compounds	IC50 (μM)
	DLD-1	MCF-7	A549
Novel 2-phenoxychromone (1)	65.8	174.4	81.9
3-O-methylquercetin (2)	16.2	16.7	34.2
3,6,4′-trimethoxychrysin (3)	17.1	50.5	19.9
3,6-dimethoxyapigenin (4)	31.0	41.9	47.0
6-methoxykaempferol (5)	101.8	>200.0	125.1
6-methoxyapigenin (6)	>200.0	>200.0	>200.0
BA propolis extract (μg/mL)	13.8	16.8	21.7

Conclusion

We identified six main compounds in ethanol extracts of BA propolis, the northeastern Brazilian propolis in the state of Bahia. One of isolated compounds was a novel 2-phenoxychromone, while the others were flavones. In vitro studies demonstrated that three 3-methoxy flavones and 2-phenoxychromone have potent growth-inhibitory activity against human colon, breast and lung cancer cell lines. This is the first report on the discovery of a novel 2-phenoxychromone from propolis.

Author contributions

K. Ichihara conducted this study, S. Tazawa prepared experimental materials, T. Mitsui and S. Hotta performed analytical and biological experiments, respectively, and T. Mitsui, Y. Arai and K. Kato analyzed the analytical data. All authors have read and approved the manuscript to be submitted.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplemental data

The supplemental data for this article can be accessed at https://doi.org/10.1080/09168451.2018.1427550

Acknowledgement

We appreciate Mr. Kazushige Abe, Nordeste Co. Ltd. (Tokyo, Japan) for supplying clump of the raw BA propolis (Special propolis). We also thank Prof. Masahiro Ebihara, Ph.D., Gifu University (Gifu, Japan) for X-ray structure determination of compound 1.