For Prof. Vassya Bankova's personal use only.
NPC
Natural Product Communications
Cytotoxicity of Constituents from Mexican Propolis Against a Panel of Six Different Cancer Cell Lines
2010 Vol. 5 No. 10 1601 - 1606
Feng Li, Suresh Awale, Yasuhiro Tezuka and Shigetoshi Kadota* Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan
[email protected] Received: December 14th, 2009; Accepted: July 6th, 2010
The cytotoxicity of 39 compounds, including eighteen flavonoids (flavanones, 1 10; flavones, 11 17; flavanol, 18), sixteen phenolic acid derivatives (aromatic acids, 19 24; aldehyde, 25; esters, 26 34) and five glycerides (35 39), isolated from Mexican propolis, were evaluated against a panel of six different cancer cell lines; murine colon 26-L5 carcinoma, murine B16-BL6 melanoma, murine Lewis lung carcinoma, human lung A549 adenocarcinoma, human cervix HeLa adenocarcinoma and human HT-1080 fibrosarcoma. A phenylpropanoid-substituted flavanol, (2R,3S)-8-[4-phenylprop-2-en-1-one]-4',7dihydroxy-3',5-dimethoxyflavan-3-ol (18), showed the most potent cytotoxicity against A549 cells (IC50, 6.2 M) and HT-1080 cells (IC50, 3.9 M), stronger than those of the clinically used anticancer drug, 5-fluorouracil (IC50, 7.5 M and 5.4 M, respectively). Based on the observed results, the structure activity relationships are discussed. Keywords: cytotoxicity, propolis, flavonoids, phenolic acid derivatives, phenylpropanoid glycerides, structure activity relationship.
Propolis is a natural hive product collected by honey bees from various plant sources [1-3]. Recently, it has been a subject of intense research, especially in the areas of oncology and a number of compounds possessing anticancer activity, such as phenethyl caffeate (CAPE), artepillin C, and propolin A C have been reported from propolis [4-10]. As a part of our study on propolis from different origins [11-17], we reported the constituents of propolis from Brazil and Myanmar, as well as their cytotoxicity against a panel of seven cancer cell lines [1821]. In our continuing study, we recently carried out a phytochemical investigation of Mexican propolis, and isolated 44 compounds. Among them, excluding minor constituents, 39 were evaluated for their cytotoxicity against a panel of six different cancer cell lines: murine colon 26-L5 carcinoma (colon 26-L5), murine B16-BL6 melanoma (B16-BL6), murine Lewis lung carcinoma (LLC), human lung A549 adenocarcinoma (A549), human cervix HeLa adenocarcinoma (HeLa), and human HT-1080 fibrosarcoma (HT-1080). Among the tested compounds, a phenylpropanoidsubstituted flavan-3-ol, (2R,3S)-8-[4-phenylprop-2-en-1one]-4',7-dihydroxy-3',5-dimethoxyflavan-3-ol (18), showed the most potent cytotoxicity against all the tested cancer cell lines with IC50 values ranging from 3.9 to 19.7 M (Table 1). Interestingly, the potencies of 18 against
A549 and HT-1080 cells were stronger than those of a positive control, 5-fluorouracil (IC50, 7.5 M for A549; 5.4 M for HT-1080). In addition, 3,3'-dimethoxy-4',5,7trihydroxyflavone (17) and 2-acetyl-3-caffeoyl-1-pcoumaroylglycerol (36) exhibited strong activity against HT-1080 cells with IC50 values of 8.4 M and 8.9 M, respectively (Tables 1 and 3), which are comparable with those of 5-fluorouracil. The other tested compounds displayed different potencies according to the cell types. Statistical significance of compounds possessing cytotoxicity at IC50 < 50 M were analyzed using a Student’s t-test and are shown in the supplementary data. Upon careful inspection of the IC50 values, structure and cytotoxic activity relationships could be deduced. In general, flavones (11 17) were found to be more active than flavanones (1 10) (Table 1). For example, 11 > 1; 12 > 2; 13 > 3; 14 > 4. These observations suggested that the presence of a double bond between C2 C3 is important for the cytotoxicity, which is in agreement with previous findings [22-25]. In flavanones, the presence of a hydroxyl group at C-3 tends to reduce the activity (4 < 1), but the presence of an O-acetyl group at C-3 increases the activity (6 > 1). Therefore, at C-3 in flavanones, the potency of cytotoxicity is in the order of O-acetyl > H > OH. Furthermore, elongation of the carbon chain length of the ester group at C-3 enhances the
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activity (10 > 9 > 7, 8). At C-7, a methoxyl group is more favorable than a hydroxyl group (1 > 2; 6 > 7). In flavones, absence of a hydroxyl group at C-4' slightly increases the activity (13 < 12). However, the role of substitution at C-7 differed among the cell types. A 7OMe group is more favorable than a 7-OH group against colon 26-L5 and HeLa cells (11 > 12; 14 > 15), but for B16-BL6, LLC, A549 and HT-1080 cells, 7-OH is more preferable than 7-OMe (11 < 12; 14 < 15).
hydroxyl group on the phenyl ring, were generally active, while other esters without a hydroxyl group were totally inactive (Table 2). Similarly, the presence of a hydroxyl group at the meta or para position on the phenyl ring is more favorable for activity than a methoxyl group (30 > 31; 33 > 32). Elongation of a double bond in the alcohol part of the ester slightly increases the activity (29 > 28). Glycerides (35−38) containing two phenylpropanoid units were active against all the tested cancer cell lines, while 39, having only one phenylpropanoid unit, was totally inactive (Table 3), suggesting an increase in the number of the phenylpropanoid unit significantly increases the activity. Similarly, on phenyl rings, an increase in the number of hydroxyl or methoxyl groups slightly enhances the activity (36 > 35; 38 > 37 > 35). Among the tested phenylpropanoid glycerides, 36 was the most active, especially against HT-1080 cells. The high potency of 36
Phenolic acid esters 29 and 33 were active against all the tested cancer cell lines (Table 2), however, their corresponding acids, 20 and 22, did not show any cytotoxicity towards any of the cell lines (IC50 > 100 µM), indicating that the presence of an ester group enhances the activity. In phenolic acid esters (26─34), a hydroxyl group on the phenyl ring contributes to the cytotoxic activity since 28, 29, 30, and 33, having a
Table 1: Structures and cytotoxicity of eighteen flavonoids (flavanones, 1─10; flavones, 11─17; flavanol, 18).
O
OH
HO
O
OCH 3 OH
OCH3
1 – 10
Colon 26-L5
Compound substitution R1 1 H 2 H 3 H 4 OH 5 OH 6 OCOCH3 7 OCOCH3 8 OCOCH2CH3 9 OCOCH(CH3)2 10 OCOCH(CH3)CH2CH3 11 H 12 H 13 H 14 OH 15 OH 16 OCH3 17 OCH3 18 5-fluorouracila doxorubicina a
positive control.
18
11 – 17
R2 OH OH OH OH OCH3 OH OH OH OH OH OH OH OH OH OH H OH
R3 OCH3 OH OH OCH3 OH OCH3 OH OH OH OH OCH3 OH OH OCH3 OH OH OH
R4
R5
H H H H H H H H H H H H H H H H OCH3
H H OH H H H H H H H H H OH H H OH OH
B16-BL6
LLC
A549
HeLa
HT-1080
IC50 (µM) 79.5 >100 >100 91.2 >100 48.0 >100 >100 91.1 71.1 7.6 76.2 88.7 30.2 74.3 >100 23.6 10.5 0.73 0.76
89.5 >100 >100 92.0 >100 60.7 >100 >100 85.1 54.1 76.5 44.8 78.8 75.6 36.1 >100 36.7 12.0 4.7 0.76
>100 >100 >100 >100 >100 >100 >100 >100 99.7 79.9 >100 98.8 >100 79.5 60.2 >100 17.5 19.7 0.97 0.73
>100 >100 >100 >100 >100 >100 >100 >100 79.8 79.3 >100 84.3 >100 >100 74.5 >100 40.5 6.2 7.5 0.88
>100 >100 >100 >100 >100 >100 >100 >100 86.3 68.1 23.6 69.5 85.3 35.6 43.6 >100 19.9 7.6 0.68 0.76
>100 >100 >100 >100 >100 72.2 >100 >100 92.7 71.2 >100 95.7 >100 >100 36.0 >100 8.4 3.9 5.4 0.53
Natural Product Communications Vol. 5 (10) 2010 1603
Cytotoxic activity of constituents from Mexican Propolis
Table 2: Structures and cytotoxicity of sixteen phenolic acid derivates (aromatic acids, 19─24; aldehyde, 25; esters, 26─34). O
O
OH
R3 R
24
2
O
R1
O
19 – 22 26 – 33
23, 25 34
Compound substitution 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 5-fluorouracila doxorubicina a
R1 H H OCH3 OCH3 H
R2 H OH OCH3 OH H
H H H H OH
OCH3 H H H H OH OCH3 OCH3 OCH3
OH H H OH OH OCH3 OCH3 OCH3 OH
H CH2Ph CH2CH=CHPh CH2Ph CH2CH=CHPh CH2CH=CHPh CH2CH=CHPh CH2Ph CH2Ph
Colon 26-L5
B16-BL6
>100 >100 >100 >100 >100 >100 >100 >100 >100 57.4 48.3 33.2 >100 >100 62.7 >100 0.73 0.76
>100 >100 >100 >100 >100 >100 >100 >100 >100 74.8 57.8 42.0 >100 >100 62.2 >100 4.7 0.76
R3
LLC A549 IC50 (µM)
HeLa
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 76.8 44.1 >100 >100 99.1 >100 0.97 0.73
>100 >100 >100 >100 >100 >100 >100 >100 >100 78.1 70.2 43.4 >100 >100 73.3 >100 0.68 0.76
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 85.2 25.3 >100 >100 95.4 >100 7.5 0.88
HT-1080 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 86.6 42.0 >100 >100 94.4 >100 5.4 0.53
positive control. Table 3: Structures and cytotoxicity of five glycerides (35─39).
35 – 38
35 36 37 38 39 5-fluorouracila doxorubicina a
Compound substitution R1 R2 H H OH H OCH3 H OCH3 OCH3
39
Colon 26-L5 47.3 29.5 41.7 31.4 >100 0.73 0.76
B16-BL6 43.6 29.3 34.8 31.1 >100 4.7 0.76
LLC A549 IC50 (µM) 62.7 79.6 28.0 43.3 54.0 79.0 32.4 49.6 >100 >100 0.97 7.5 0.73 0.88
HeLa
HT-1080
63.6 21.7 51.9 38.3 >100 0.68 0.76
73.7 8.9 69.2 33.4 >100 5.4 0.53
Positive control.
might be attributed to the presence of a catechol moiety, which has the capacity to induce cytotoxic activity via its strong anti-oxidant behavior [26]. In conclusion, 39 compounds, isolated from Mexican propolis, were evaluated for their cytotoxicity against a panel of six different cancer cell lines and
established the structure-activity relationship. (2R,3S)8-[4-Phenylprop-2-en-1-one]-4',7-dihydroxy-3',5dimethoxyflavan-3-ol (18), cinnamyl p-coumarate (29) and 2-acetyl-3-caffeoyl-1-p-coumaroylglycerol (36) displayed the most potent cytotoxicity among the tested flavonoids, phenolic acid derivatives and glycerides, respectively.
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Experimental Chemicals: Eighteen flavonoids: pinostrobin (1), pinocembrin (2), naringenin (3), alipinone (4), pinobanksin 5-methyl ester (5), alipinone 3-acetate (6), pinobanksin 3-acetate (7), pinobanksin 3-propanoate (8), pinobanksin 3-isobutyrate (9), pinobanksin 3-(2methyl)-butyrate (10), tectochrysin (11), chrysin (12), apegenin (13), izalpinin (14), galangin (15), 3-methoxy4',7-dihydroxyflavone (16), 3,3'-dimethoxy-4',5,7trihydroxyflavone (17), and (2R,3S)-8-[4-phenylprop2-en-1-one]-4',7-dihydroxy-3',5-dimethoxyflavan-3-ol (18), and sixteen phenolic acid derivatives: cinnamic acid (19), p-coumaric acid (20), 3,4-dimethoxycinnamic acid (21), ferulic acid (22), benzoic acid (23), cinnamylideneacetic acid (24), vanillin (25), benzyl cinnamate (26), cinnamyl cinnamate (27), benzyl pcoumarate (28), cinnamyl p-coumarate (29), cinnamyl isoferulate (30), cinnamyl 3,4-dimethoxycinnamate (31), benzyl 3,4-dimethoxycinnamate (32), benzyl ferulate (33), and cinnamyl benzoate (34), together with five glycerides: 2-acetyl-1,3-di-p-coumaroylglycerol (35), 2-acetyl-3-caffeoyl-1-p-coumaroylglycerol (36), 2-acetyl-1-p-coumaroyl-3-feruloylglycerol (37), 2acetyl-1,3-diferuloylglycerol (38), and 3-acetyl-1-pcoumaroylglycerol (39), were isolated from the MeOH extract of Mexican propolis. The identities of these compounds were determined by analyzing their spectroscopic data and confirmed by comparing their values with those in the literature. Cancer cell lines and culture conditions: Highly liver metastatic murine colon 26-L5 carcinoma cell line, highly liver metastatic murine B16-BL6 melanoma cell line, and highly lung metastatic murine Lewis lung carcinoma cell line were obtained from Dr I. Saiki (Institute of Natural Medicine, University of Toyama), Dr I. J. Fidler (M. D. Anderson Cancer Center, Houston, TX, U.S.A.) and Dr K. Takeda (Juntendo University, Tokyo), respectively and maintained in our laboratory. Highly metastatic human HT-1080 fibrosarcoma cell line (ATCC#CCL-121) was obtained from American Type Culture Collection (Rockville, MD, U.S.A.). Human lung A549 adenocarcinoma (RCB0098) and human cervix HeLa adenocarcinoma (RCB0007) cell lines were purchased from Riken Cell Bank (Tsukuba, Japan). All the cancer cell lines were maintained in -modified minimum essential medium (MEM , Wako Pure Chemicals Ind., Ltd., Osaka, Japan), except for murine colon 26-L5 carcinoma cell line, which was maintained in RPMI 1640 medium (Wako Pure Chemicals Ind., Ltd., Osaka, Japan). Both of these media were supplemented with 10% fetal bovine serum (FBS; Gibco BRL Products, Gaithersburg, MD), 0.1% sodium bicarbonate (Nacalai Tesque, Inc., Kyoto, Japan) and 1% antibiotic
Li et al.
antimycotic solution (Sigma-Aldrich Inc., St. Louis, U.S.A.). Cytotoxic activity assay: Cell viability other than LLC, in the presence or absence of tested compounds, was determined using the standard 3-(4,5-dimethyl- thiazol2-yl)-2,5-dimethyltetrazolium bromide (MTT; SigmaAldrich Inc., St. Louis, U.S.A.) assay [27], as described previously [11]. In brief, exponentially growing cells were harvested and 2×103 cells suspended in 100 L of medium per well were plated in a 96-well plate (Corning Inc., NY, U.S.A.). After 24 h incubation at 37°C under a humidified 5% CO2 to allow cell attachment, the cells were treated with varying concentrations of test specimens in their respective medium (100 L) and incubated for 72 h under the same conditions. Two h after the MTT (0.5 mg/mL, 100 L/well) addition, the formazan formed was extracted with dimethyl sulfoxide (DMSO, 100 L/well, Wako Pure Chemicals Ind., Ltd., Osaka, Japan) and its amount measured spectrophotometrically at 550 nm with a Perkin-Elmer HTS-7000 Bio Assay Reader (Norwalk, CT, U.S.A.). In the case of LLC cells, a standard crystal violet staining (Nacalai Tesque, Inc., Kyoto, Japan) assay was used, following the literature procedure [28]. In brief, exponentially growing cells were harvested and 1×103 cells, suspended in 100 L of medium per well, were plated in a 96-well plate. After 24 h of incubation at 37°C under a humidified 5% CO2 atmosphere, 100 L of medium containing various concentrations of test specimen was added to each well and incubated for 72 h under the same conditions. After fixation with 25% glutaraldehyde solution (20 L, Wako Pure Chemicals Ind., Ltd., Osaka, Japan), the cells were stained with 0.5% crystal violet in 20% methanol/water for 30 min. After gentle rinsing with water, the retained crystal violet was extracted with 30% acetic acid (Wako Pure Chemicals Ind., Ltd., Osaka, Japan) and measured spectrophotometrically at 590 nm. Each compound was dissolved by DMSO and then diluted with medium. The final concentration of DMSO was less than 0.5%, which had no effect on the cancer cells. 5-Fluorouracil (Tokyo Kasei Kogya Co. Ltd., Tokyo, Japan) and doxorubicin (Kyowa Hakko Co. Ltd., Tokyo, Japan) were used as positive controls, and IC50 values (the concentration of compound producing 50% cell growth inhibition relative to untreated control) were calculated from the mean values of data from 3 wells. Acknowledgments - This work was supported by a grant from the Ministry of Health and Welfare for the Third-Term Comprehensive 10-Year Strategy for Cancer Control.
Cytotoxic activity of constituents from Mexican Propolis
Natural Product Communications Vol. 5 (10) 2010 1605
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