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Antiproliferative Activity of Flavonoids from Croton sphaerogynus Baill. (Euphorbiaceae)
Croton sphaerogynus is a shrub from the Atlantic Rain Forest in southeastern Brazil. A lyophilized crude EtOH extract from leaves of C. sphaerogynus, obtained by maceration at room temperature (seven days), was suspended in methanol and partitioned with hexane. The purified MeOH phase was fractionated over Sephadex LH-20 yielding five fractions (F1–F5) containing flavonoids, as characterized by HPLC-DAD and HPLC-MS analyses. The antiproliferative activity of the crude EtOH extract, MeOH and hexane phases, and fractions F1–F5 was evaluated on in vitro cell lines NCI-H460 (nonsmall cell lung), MCF-7 (breast cancer), and U251 (glioma). The MeOH phase showed activity (mean log GI50 0.54) higher than the hexane phase and EtOH extract (mean log GI50 1.13 and 1.19, resp.). F1 exhibited activity against NCI-H460 (nonsmall cell lung) (GI50 1.2 μg/mL), which could be accounted for the presence of flavonoids and/or diterpenes. F4 showed moderate activity (mean log GI50 1.05), while F5 showed weak activity (mean log GI50 1.36). It is suggested that the antiproliferative activity of the crude EtOH extract and MeOH phase is accounted for a synergistic combination of flavonoids and diterpenes.
Croton L. (Euphorbiaceae) has approximately 1300 species of herbaceous, shrubs, trees, and lianas forms. The genus is widely distributed in tropical and subtropical regions around the world, including Brazil, a country with 316 species, among which 253 are endemic [1, 2].
Medicinal and toxic properties of Croton species have been ascribed to a wide variety of chemical compounds, such as terpenoids and steroids, alkaloids, and phenolic compounds, the latter including predominantly flavonoids, lignans, and proanthocyanidins [3–5].
According to the International Agency for Research on Cancer (IARC), the world impact of cancer has more than duplicated in the last 30 years . The 2014 and 2015 annual estimative regarding Brazil foresees the emergence of approximately 576,000 new cancer cases, including nonmelanoma skin cancer. This latter cancer type is expected to become the most frequent among the Brazilian population (32% new cases), followed by prostate tumors (12%), female breast (10%), colon and rectum (6%), lung (5%), stomach (3.5%), and cervical (3%) .
Several studies dealing with Croton species have reported the isolation of cytotoxic derivatives. Shoots of C. hieronymi Griseb. showed strong activity against lung A-549 carcinoma cells, mouse lymphoma, and human colon carcinoma . The CH2Cl2 extract of leaves of C. macrobothrys Baill. and C. zambesicus Müll. Arg. showed cytotoxicity against human lung and leukemia cells  and cervix carcinoma cells , respectively. Ent-kaurane diterpenoids from Croton tonkinensis have activity against colorectal cancer cells ; ent-kaur-16-ene-6a,19-diol from C. floribundus Spreng. exhibited a moderate effect against MDA-MB-435 (melanoma), HCT-8 (colorectal adenocarcinoma), and HCT-116 (colorectal adenocarcinoma) cell lines . Isopimara-7,15-dien-3b-ol from C. zambesicus showed weak cytotoxic activity against cancer (HeLa, human cervix carcinoma cells; HL-60, human promyelocytic leukemia) and noncancer (WI-38, human lung fibroblast) cell lines . An epimer of kaurenoic acid from C. antisyphiliticus Mart. showed cytotoxic activity, with half maximal inhibitory concentration values of 59.41, 68.18, and 60.30 g/mL for the B-16 (murine melanoma), HeLa (human cervix carcinoma cells), and 3T3 (normal mouse embryo fibroblasts) cell lines, respectively . These compounds are considered valuable toward the development of new and highly effective anticancer chemotherapeutic agents, due to their efficacy toward the induction of apoptosis [17, 18].
Individuals of Croton sphaerogynus Baill. sect. Cleodora grow in the states of Bahia, Rio de Janeiro, Espírito Santo, and São Paulo (Brazil). Most of its populations are distributed in moist forests of seashore plains (“restinga forests”). Two other species of this section, Croton cajucara Benth., popularly known as “sacaca,” and Croton heterocalyx Baill., have been traditionally exploited for their medicinal properties (e.g., [4, 5, 19]). CH2Cl2 and hexane extracts from leaves of C. sphaerogynus exhibited antiproliferative activity against NCI-H460 (nonsmall cell lung) (GI50 0.26 g/mL and 0.33 g/mL, resp.) and K562 cell lines (GI50 0.60 g/mL and <0.25 g/mL, resp.). These activities were related to the presence of abietane, podocarpane, and clerodane diterpenes .
The main goal of this study was to characterize the major polar constituents of the EtOH extract from leaves of C. sphaerogynus and evaluate their in vitro antiproliferative activities against tumor cell lines. This study differs from that of Motta et al. , which focused on the antiproliferative activity of diterpenes of nonpolar extracts from C. sphaerogynus and thus did not deal with its flavonoid constituents and their antiproliferative activity.
2. Material and Methods
2.1. Plant Material
Leaves of Croton sphaerogynus were collected in January 2012, in an area of Atlantic Forest in the municipality of Itanhaém, State of São Paulo (Southeastern Brazil). A voucher specimen (LBM 65) was deposited in the herbarium Maria Eneyda P. K. Fidalgo (SP), São Paulo.
2.2. Extraction Procedure
Dried and powdered leaf material (1 kg) was extracted by maceration with EtOH (5 L) for seven days at room temperature. Crude EtOH extract (EE) was concentrated under reduced pressure, evaporated to dryness under a stream of nitrogen, and lyophilized, affording 70.62 g of crude EtOH extract (yield: 7%). Part of the crude EtOH extract (65 g) was resuspended in MeOH and partitioned using hexane. The hexane phase (HP) was concentrated under reduced pressure to yield 20 g (2%) of hexane phase, while the MeOH phase (MP) was lyophilized to afford 15 g (yield: 1.5%). Part of MP (8 g) was fractionated over Sephadex LH-20 using MeOH as eluent, affording five fractions: F1 (2 g), F2 (0.119 g), F3 (0.090 g), F4 (0.353 g), and F5 (0.090 g).
2.3. Chemical Analysis
All lyophilized crude EtOH extract, MP and HP phases, and fractions (F1–F5) were dissolved in MeOH (2 mg/mL) and analyzed using a HPLC 1260 (Agilent Technologies) chromatograph, equipped with diode array detector (DAD) and a Zorbax-C18 ( mm, 3.5 m, Agilent, USA) column at 40°C. Solvents used were 0.1% acetic acid (AcOH) and acetonitrile (CH3CN), starting with 15% of CH3CN (0–20 min), increasing to 100% (20–25 min), isocratic (5 min), and decreasing to 15% (30–32 min), and isocratic (3 min). Solvent flow rates were 1.5 mL/min (0–25 min), 1.0 mL/min (25-26 min), and 1.5 mL/min (26–35 min); injection volume was 3 L, and detection was at 352 nm and 280 nm. Quercetin and kaempferol at concentrations from 0.6 to 360 ng/mL were used to prepare calibration curves following the same analysis conditions. Results are expressed in milligrams per gram of dry sample (mg/g).
MeOH phase (MP) was also analyzed using a Bruker Daltonics equipment Esquire 3000 Plus HPLC with a Zorbax-C18 ( mm, 3.5 μm, Agilent, USA) column at 40°C, using the same conditions cited above. Solvent flow rates was .90 L/min, voltage 4000 V, nebulizer 27 psi, drying gas at 320°C, and flow of 7 L/min. Constituents were identified by comparing the corresponding UV-Vis and ESI/MS-MS spectra with MS data from the literature.
A purified compound from F5 was analyzed by 1H NMR at 500 MHz, using a Bruker DRX-500 spectrometer. DMSO-d6 (Aldrich) was used as solvent and the residual peak of the nondeuterated solvent as internal standard.
2.4. Antiproliferative Assay
Cancer cell lines used were kindly provided by the National Cancer Institute (NCI) at Frederick MA-USA and included NCI-H460 (nonsmall cell lung), MCF-7 (breast cancer), and U251 (glioma). Stock cell cultures were grown in medium containing RPMI 1640, supplemented with 5% of fetal bovine serum. Experimental cultures were supplemented also with penicilin : streptomicin (10 g/mL : 10 UI/mL).
Cells (100 L cells/well, inoculation density from 3–6 104 cell/mL) in 96-well plates were exposed to various sample concentrations (0.25 to 250 g/mL, 100 L/well) in DMSO/RPMI 1640/FBS 5% at 37°C, 5% of CO2 in air for 48 h. Final DMSO concentration did not affect cell viability. Cells were then fixed with 50% trichloroacetic acid and cell proliferation was determined by spectrophotometric quantification of cellular protein content at 540 nm, using the sulforhodamine B assay. Doxorubicin (DOX; 0.025–25 g/mL) was used as positive control. Three measurements were obtained at the beginning of incubation (time zero, ) and 48 h after incubation for compound-free () and tested () cells. Cell proliferation was determined according to the equation , for , and , for and a concentration-response curve for each cell line was plotted using software ORIGIN 7.5 (OriginLab Corporation) .
2.5. Data Analysis
Using the concentration-response curve for each cell line, GI50 (concentration causing 50% growth inhibition)  was determined by means of nonlinear regression analysis, using software ORIGIN 7.5 (OriginLab Corporation). The average activity (mean of log GI50) of the extracts tested was also determined using MS Excel software. Extracts were regarded as inactive (mean > 1.5), weakly (1.1 < mean < 1.5), moderately (0 < mean < 1.1), or potently (mean < 0) active on basis of the NCI criteria for the mean of log GI50 .
3. Results and Discussion
Retention times and UV and MS data analysis of the constituents from the crude EtOH extract (EE), MeOH (MP) and hexane (HP) partition phases, and fractions F1–F5 are given in Table 1. Twenty substances were detected, mainly quercetin and kaempferol derivatives. F1, F2, and F3 exhibited similar flavonol composition. F4 contains three major compounds: quercitrin, quercetin 3-O-methyl ether, and a kaempferol derivative, while F5 contains quercetin 3-O-methyl ether (Table 1, compound 17).
|Rt: average retention time in minutes. Compound confirmed by 1H NMR.|
Flavonoids found in Croton are mostly highly methoxylated aglycones, such as artemetin . It has been reported that several Croton species produce acylated flavonoids, such as tiliroside (kaempferol--coumaroyl glucoside) [25–27]. Rutin (Table 1, compound 7) was also detected in the MeOH extract from leaves of C. lechleri Mull. Arg. ; fresh latex of C. celtidifolius Baill. has flavonols, such as kaempferol, quercetin, and myricetin in its composition . Savietto et al.  detected apigenin dihexoside and tiliroside (kaempferol-p-coumaroyl glucoside) as ubiquitous constituents of the MeOH leaf extract of C. dichrous Müll. Arg., C. erythroxyloides Baill., and C. myrianthus Mull. Arg.
C. sphaerogynus was previously described as a major producer of diterpenes. Using serial extraction with hexane, CH2Cl2, and MeOH, Motta et al.  identified a great diversity of diterpenes, especially in the CH2Cl2 extract. In the present study, maceration with EtOH at room temperature yielded an extract with a diterpene profile similar to that described by Motta et al. . The lyophilized crude ethanol extract was resuspended in MeOH and partitioned with hexane. Partition did not eliminate the diterpenes from the polar fraction MP. Column chromatography using Sephadex and MeOH gave five fractions (F1–F5), the first of which (F1) contained diterpenes and flavonoids, while the further fractions (F2–F5) contained flavonoids exclusively.
Crude EtOH extract(EE), hexane phase (HP), F1, and F2 showed weak antiproliferative activity (Table 2). Methanol phase (MP) and F4 showed moderate activity, mainly against cell lines NCI-H460 (nonsmall cell lung) (mean log GI50 0.54 and 1.05, resp.). On the other hand, F3 was inactive, while F5, containing virtually only quercetin-3-O-methyl ether (Table 1, compound 17), also showed weak antiproliferative activity (Table 2).
|U251: glioma; MCF-7: breast cancer; NCI-H460: nonsmall cell lung.|
bPositive control. NCI’s criteria (Fouche et al., 2008 ): I: mean log GI50 > 1.5 = inactive; W: weak activity: mean log GI50 > 1.1–1.5; M: moderate activity: mean log GI50 > 0–1.1; P: potent activity: mean log GI50 < 0.
Crude EtOH extract (EE); MeOH (MP) and hexane (HP) phases, and fractions (F1, F2, F3, F4, and F5).
According to Motta et al.  the antiproliferative activity of C. sphaerogynus extract was a result of the massive presence of abietane and/or podocarpane diterpenes in nonpolar extracts. The present study tested two different sets of samples: extract and phases composed by different proportions of diterpenes and flavonoids (EE, HP, MP,and fraction F1) and fractions composed exclusively by flavonoids (F2–F5). Table 3 compares data obtained by Motta et al.  and the diterpene mixture obtained in the present work. According to Motta et al.  the CH2Cl2 extract showed higher activity (mean log GI50 0.86), compared with hexane and MeOH extracts (mean log GI50 1.26 and 1.49, resp.). Regarding the diterpene profile, the latter extract was the most similar to MP and HP phases, although some qualitative and quantitative differences are evident. No crotonin derivative was detected in the present study, which may explain the moderate antiproliferative activity of the CH2CL2 extract (mean 0.86 log GI50) reported by Motta et al.  and the weak antiproliferative activity exerted by HP. The CH2Cl2 extract from C. macrobothrys, which contains a crotonin derivative, showed moderate antiproliferative activity (mean log GI50 0.89) . Grynberg et al.  tested trans-dehydrocrotonin and trans-crotonin isolated from C. cajucara Benth. on the survival of mice bearing Sarcoma 180 and Ehrlich carcinoma and observed a significant antitumor activity when mice were treated with trans-dehydrocrotonin.
|Main constituents of leaf extract of Croton sphaerogynus and respective GC/EIMS data and characterization according to Motta et al. . Dch, CH2Cl2; Met, MeOH.|
MP phase was the most active sample, showing moderate antiproliferative activity. The relative proportion of diterpenes and flavonoids (Table 4) might be important to enhance the antiproliferative activity. Either extracts with high contents of diterpenes or fractions with high contents of flavonoids presented weak or no antiproliferative activity. F2, F3, and F4 (fractions lacking diterpenes) were shown to be inactive. F1, though still containing diterpenes, showed flavonols in smaller amount than MP; F5, composed virtually by quercetin-3-O-methyl ether, also showed weak antiproliferative activity. The hexane phase (HP) contained no detectable flavonoids. These results suggest that Croton species lacking crotonin derivatives might have moderate antiproliferative activity if they have a combination of other diterpenes and flavonols.
|Identification suggestions in Table 1.|
QValues expressed as milligram per gram of quercetin equivalent (mg/g QE).
KValues expressed as milligram per gram of kaempferol equivalent (mg/g KE).
nd: trace amounts.
Extracts from Croton species are frequently reported as exerting antiproliferative activity. The essential oil from the stem bark of C. lechleri showed mutagen-protective efficacy  and crude extracts from stems of C. cajucara , containing clerodane diterpenes, exert antitumor activity against the K562 leukemic cell line. The CH2Cl2 extract of C. macrobothrys leaves, also containing clerodane diterpenes, exhibited moderate antiproliferative activity against several cell lines, in particular NCI-H460 (nonsmall cell lung) and K562 (leukemia) .
On the other hand, flavonoids such as apigenin dihexoside and tiliroside (kaempferol-p-coumaroyl glucoside) detected in leaf extract of C. dichrous, C. myrianthus, and C. erythroxyloides showed weak or no growth cell inhibition. However, extracts or fractions with substantial amounts of these compounds showed weak activity . However, MeOH extract from C. erythroxyloides obtained under reflux showed moderate antiproliferative activity (mean of log GI50 1.00) .
Angst et al.  observed that the flavonol quercetin inhibits the growth of pancreatic cancer cell lines by inducing apoptosis. The association of gemcitabine (a standard chemotherapeutic drug administered to patients with pancreatic cancer) and quercetin had no additional effect when compared with quercetin administered alone. The authors also observed a significant apoptotic effect and reduced tumor cell proliferation in in vivo assay using quercetin.
Besides synergism, the chemical structure of the flavonoids seems to be directly related to their antiproliferative activity. Burmistrova et al.  showed that synthetic flavonols with a hydroxyl group at the C3 position are 7-fold more potent than flavonols with a methoxyl group at the same position. This result suggests that a C3 methoxyl group at C3 is a reducing factor of cytotoxicity. In the present study, quercetin-3-O-methyl ether alone showed weak antiproliferative activity (mean of log GI50 1.36). However, according to Seito et al.  flavonoids with methoxyl groups at positions other than C3 seem to have inhibitory effect on cell growth.
The antiproliferative activity of Croton sphaerogynus seems to be related to the presence of diterpenes and flavonoids. MeOH phase (MP) presented the highest antiproliferative activity among all samples tested and is showed to be composed by diterpenes and a high amount of flavonoids, in comparison with the crude EtOH extract (EE) and F1. Fractions containing no diterpenes showed weak antiproliferative activity. Samples containing small proportions of flavonoids also showed weak antiproliferative activity. The relative proportions of representatives of these two metabolite classes (flavonoids and diterpenes) in C. sphaerogynus extracts seem to be crucial to determine their antiproliferative activity.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support (FAPESP 2012/10079-0) and Frederick Cancer Research & Development Center of the National Cancer Institute (Frederick, MA, USA) for the kind provision of cell lines. Antonio Salatino, Cláudia M. Furlan, Deborah Y. A. C. Santos, João E. de Carvalho, João Henrique G. Lago, Marcelo J. Pena Ferreira, and Maria L. F. Salatino are fellow researchers of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Kátia Pereira Santos thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a Master’s research grant.
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