PHYTOTHERAPY RESEARCH Phytother. Res. 20, 701–703 (2006) Published online 4 May 2006 in Wiley InterScience ANTIPROLIFERATIVE EFFECTS OF SALVIA (www.interscience.wiley.com) DOI: 10.1002/ptr.1911
701
SHORT COMMUNICATION
In vitro Antiproliferative Effect of Six Salvia species on Human Tumor Cell Lines Giovina Fiore1, Cristina Nencini1, Federica Cavallo2, Anna Capasso2, Ammar Bader3, Giorgio Giorgi1 and Lucia Micheli1* 1
Department of Pharmacology ‘Giorgio Segre’, University of Siena, Siena, Italy Department of Pharmaceutical Sciences, University of Salerno, Italy 3 Faculty of Pharmacy, Al-Zaytoonah Private University of Jordan, Amman, Jordan 2
This study was designed to examine the in vitro antiproliferative activity of the methanol crude extracts of six Salvia species: Salvia dominica L. leaves, Salvia lanigera Desf. aerial parts, Salvia menthaefolia Ten. roots, Salvia palaestina Benth. aerial parts, Salvia sclarea L. roots and Salvia spinosa L. aerial parts. Extracts were screened for their possible antitumoral activity by MTT test on nine human cancer cell lines: glioblastoma (DBTRG-05MG, T98G, U-87MG), colorectal adenocarcinoma (WiDr and HT-29), prostate adenocarcinoma (MDA Pca2b), choriocarcinoma (JEG-3), endometrium adenocarcinoma (HEC-1A) and B lymphoblast (CIR). IC50 values were determined for only five extracts and ranged from 90 to 400 µg/mL approximately. Salvia menthaefolia extract exhibited marked antiproliferative activity against all tumor cell lines showing lower IC50 values, while S. spinosa, S. sclarea and S. dominica extracts showed a degree cytotoxic activity dependent on the cell line type. Finally S. palaestina extract revealed a moderate antiproliferative effect only against three cell lines. Salvia lanigera extract displayed toxic activity at all concentrations tested. The results strengthen the evidence that the genus Salvia could be considered a natural resource of potential antitumor agents. Copyright © 2006 John Wiley & Sons, Ltd. Keywords: Salvia species; extracts; antitumoral activity; cell lines; toxicity.
INTRODUCTION The genus Salvia belongs to the Lamiaceae family, comprises numerous species and shows diverse biological activities manifested by the different components, that allow for the many medicinal and pharmaceutical applications of the plant materials and/or extracts. In fact, many diterpenes, isolated from plants of several species of the genus Salvia, have been demonstrated to have interesting pharmacological properties, such as antioxidant (Lima et al., 2005), antimicrobic (Gonzales et al., 1987), antiinflammatory, analgesic (Hosseinzadeh et al., 2003), antipyretic, hemostatic (HernandezPerez et al., 1995), hypoglycemic (Alarcon-Aguilar et al., 2002) and antitumor (Liu et al., 2000). Indeed, Salvia miltiorrhiza Bunge, commonly used in traditional Chinese herbal medicine to treat cardiovascular and hepatic disorders, has been shown recently to have a significant antitumoral property (Chang et al., 2004). Moreover a diterpenequinone (4,5-seco-5,10-friedoabieta-3,5(10),6,8,13-pentaene-11,12-dione) isolated from Salvia prionitis Hance exhibited not only antibacterial, antitubercular and antiphlogistic activity but even cytotoxic property against cell lines in vitro (Zhang et al., 1999).
* Correspondence to: Professor Lucia Micheli, Department of Pharmacology ‘Giorgio Segre’, University of Siena, Via Delle Scotte 6, 53100 Siena, Italy. E-mail:
[email protected] Contract/grant sponsor: PAR (Athenaeum plan for the research). Copyright © 2006 John Wiley & Sons, Ltd. Copyright © 2006 John Wiley & Sons, Ltd.
Successively this natural product has been modified structurally to give a novel compound, named salvicine, that displayed a marked antitumor activity on human solid tumor cell lines (Qing et al., 2001). Salvicine was considered a promising compound to develop a new anticancer drug, and it has been chosen for preclinical studies. On the basis of these findings, Salvia species could be considered as a source of potential antitumor agents. The aim of this study was to screen the antiproliferative activity of several Salvia species. Methanol crude extracts of six Salvia species: Salvia dominica L. leaves, Salvia lanigera Desf. aerial parts, Salvia menthaefolia Ten. roots, Salvia palaestina Benth. aerial parts, Salvia sclarea L. roots and Salvia spinosa L. aerial parts were evaluated for their antiproliferative activity on nine human cancer cell lines: glioblastoma (DBTRG, T98, U87), colorectal adenocarcinoma (WiDr and HT29), prostate adenocarcinoma (MDA), choriocarcinoma (JEG3), endometrium adenocarcinoma (HEC-1A) and B lymphoblast (CIR).
MATERIALS AND METHODS The leaves of Salvia dominica L. were collected in AsSalt (Jordan) in April 2003, the aerial parts of Salvia lanigera Poiret. were collected in Al-Rusaifeh near Zarqa (Jordan) in June 2003, the aerial parts of Salvia palaestina Benth. were collected in Amman (Jordan) and the aerial parts of Salvia spinosa L. were collected in Al-Hashemiyyeh (Jordan), in April 2003. All these Received 6 March(2006) 2006 Phytother. Res. 20, 701–703 Accepted 10 March 2006 DOI: 10.1002/ptr
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G. FIORE ET AL.
Jordanian Salvia species were identified by Dr. Ammar Bader, Al-Zaytoonah Private University of Jordan and their voucher specimens (Jo-It-2003/3; Jo-It-2003/1; JoIt-2003/2; Jo-It-2003/4; respectively) are deposited in the Herbarium of the Laboratory of Pharmacognosy and Phytochemistry at Al-Zaytoonah Private University, Jordan. The roots of Salvia menthaefolia Ten. were collected in the Botanical Garden of Palermo, in April 2002, identified by Prof. G. Venturella, University of Palermo and deposited in the Herbarium of the Botanical Garden of Palermo. The roots of Salvia sclarea L. were supplied by INDENA S.p.A. Extraction. Dried and powdered parts of each Salvia species (50 g) were pretreated with n-hexane and successively extracted (500 mL × 3 times) with methanol. The extract solutions were filtered and concentrated in vacuum, obtaining crude dried methanol extracts (Table 1). Cell lines and culture. The following human cancer cell lines were used for this study: WiDr and HT29 (colorectal adenocarcinoma), T98G (glioblastoma multiforme), DBTRG-05MG (glioblastoma), U-87MG (glioblastoma, astrocytoma), HEC-1-A (endometrium adenocarcinoma), JEG-3 (choriocarcinoma), MDA Pca 2b (prostate adenocarcinoma) and CIR (B lymphoblast). All cell lines used were obtained from the American Type Culture Collection (ATCC, Rockville MD). HT29, T98G, U-87 MG, HEC-1-A, JEG-3 cells were grown in DMEM supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% heat-inactivated fetal bovine serum. DBTRG05MG, WiDr, MDA PCa 2b and CIR cells were grown in RPMI supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum. All cell lines were maintained in a humidified 5% CO2–95% air at 37 °C. Antiproliferative assay. The cells were plated in 96well flat bottom plates at 5000 cell/well and were allowed to adhere to the wells overnight, then the cells were Table 1. Salvia species tested for growth inhibitory activity
Salvia species S. S. S. S. S. S.
menthaefolia sclarea dominica spinosa palestina lanigera
Plant part
Extract yield (mg/g dried salvia)
Roots Roots Leaves Aerial parts Aerial parts Aerial parts
126 118 163 175 183 129
treated with different concentrations of the crude Salvia extracts (0 –500 µg/mL) with a final DMSO concentration of 1%. After 72 h, a tetrazolium dye colorimetric test, MTT, was used to monitor cell growth (Hansen et al., 1989). The absorbance of converted dye was measured at a wavelength of 570 nm with background subtraction at 630 nm. Each experiment used as a positive control, cells treated with a well known antitumoral compound, mitomycin C 1 µM. Mitomycin C concentration was obtained determining IC50 value by dose-response curve. Data analysis. Data were expressed as the percent variation with respect to the control (each point resulted by three independent experiments, performed in triplicate). The percentages of cell growth were used to obtain the full dose response curves and to determine the IC50 values (concentration inhibiting of 50% the cell growth compared with control).
RESULTS AND DISCUSSION Six crude methanol extracts were screened in human cancer cell lines from different histological types (brain, colon, prostate, uterus, placenta and peripheral blood). Extracts were tested for growth inhibitory activity at a range of 0 –500 µg/mL after 72 h of treatment. Full dose-response curves were then obtained for only five extracts that demonstrated greater than 50% growth inhibition. IC50 values, shown in Table 2, were determined from these dose response curves. The Salvia lanigera extract did not show a dose response effect but displayed toxic activity at all concentrations tested in each cell line. Comparing the different species, Salvia menthaefolia extract exhibited marked antiproliferative activity against all tumor cell lines with lower IC50 values. Moreover S. spinosa, S. sclarea and S. dominica extracts showed a degree cytotoxic activity dependent on the cell line type, and S. palaestina extract displayed a moderate antiproliferative effect only against three cell lines (HEC-1A, JEG-3, CIR). The data obtained suggest that there are great differences among the various species, and the results strengthen the evidence that the genus Salvia could be considered a natural resource of antitumor agents. Acknowledgements Authors would like to acknowledge N. De Tommasi (Department of Pharmaceutical Sciences, University of Salerno, Naples, Italy) for the extracts. This work was supported by PAR (Athenaeum plan for the research).
Table 2. In vitro growth inhibitory activity (IC50 µg/ml) of crude methanolic extracts from different Salvia species Salvia species
WiDr
HT-29
DBTRG-05MG
T98G
U-87MG
HEC-1A
JEG-3
MDA Pca2b
CIR
S. S. S. S. S.
89.6 194.4 175.5 152.8 –
95.3 196.1 119.5 129.4 –
188.3 402.8 308.2 405.1 –
314.5 369.1 – 333.6 –
238.0 – – – –
141.4 215.4 192.5 151.2 404.2
108.9 234.8 114.2 120.7 377.6
339.3 294.4 405.9 299.2 –
137.1 147.6 324.3 154.5 292.3
menthaefolia sclarea dominica spinosa palestina
Copyright © 2006 John Wiley & Sons, Ltd.
Phytother. Res. 20, 701–703 (2006) DOI: 10.1002/ptr
ANTIPROLIFERATIVE EFFECTS OF SALVIA
703
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Copyright © 2006 John Wiley & Sons, Ltd.
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Phytother. Res. 20, 701–703 (2006) DOI: 10.1002/ptr
