Abstract
Total extracts of the aerial parts of four Haplophyllum. species (Rutaceae) including Haplophyllum stapfianum. Hand.-Mazz., H. tuberculatum. (Forssk.) Adr. Juss., H. acutifolium. (DC.) G. Don, and H. viridulum. Soják were tested for in vitro. cytotoxicity against 11 tumor cell lines and normal peripheral blood mononuclear cells (PBMCs). Cytotoxicity of Haplophyllum. species were also compared with Ruta graveolens. L. (Rutaceae) extract. Cytotoxic activity of extracts was evaluated by the WST-1 assay and expressed as IC50 values. The results obtained indicated that among hematopoietic tumor cell lines, strong cytotoxic activity was observed from H. stapfianum., H. tuberculatum., and H. acutifolium. against RAMOS cell line with IC50 values of 12.3, 25.3, and 23.7 µg/mL, respectively; H. stapfianum. and H. tuberculatum. against U937 cell line with IC50 values of 15.6 and 29.3 µg/mL; and H. tuberculatum. against RPMI-8866 cell line with IC50 = 31.8 µg/mL. Testing plant extracts on solid tumor cell lines revealed that all Haplophyllum. species have markedly high cytotoxicity against LNCap-FGC-10, a prostate adenocarcinoma cell line, except H. viridulum.. H. tuberculatum. also showed strong cytotoxic effect against 5637, a bladder carcinoma cell line (IC50 = 23.3 µg/mL). Investigating cytotoxic activity of R. graveolens. resulted in higher IC50 values against all tumor cell lines except LNCap-FGC-10 (IC50 = 27.6 µg/mL). Cytotoxicity evaluation of Haplophyllum. species on PBMCs revealed that resulting IC50 values for H. stapfianum. and H. acutifolium. on tumor cell lines with high cytotoxic activity were significantly lower than the IC50 values obtained for PBMCs (p < 0.05).
Introduction
The genus Haplophyllum. (Rutaceae) has about 70 species, which are distributed throughout warm temperate and subtropical zones of Eurasia and the northern tropical zone of eastern Africa (Townsend, Citation1986). Iran, with 25 Haplophyllum. species of which 13 are endemic, has the highest number of Haplophyllum. species and can be called a center of speciation of this genus (Townsend, Citation1986).
A chemical literature survey reveals the presence of lignans of diverse structure (Gozler et al., Citation1994aCitation1994bCitation1996; Evcim et al., 1986), coumarins (Ulubelen et al., Citation1993), sterols, flavonoids (Yuldeshev, Citation2001Citation2002), and several classes of alkaloids (Gozler et al., Citation1996; Sheriha et al., Citation1987; Ulubelen et al., Citation1993; Puricelli et al., Citation2002) in different species of the genus Haplophyllum.. Many biological activities have been reported from various Haplophyllum. species and include antitumor effects of H. dauricum. (L.) G. Don (Graham et al., Citation2000), cytotoxicity of five lignans of H. patavinum. (L.) G. Don against LoVo human colon carcinoma cells (Innocenti et al., Citation2002), and cytotoxic effect of H. tuberculatum. (Forssk.) Adr. Juss. on various strains of Plasmodium falciparum. (Khalid et al., Citation1986). There are also reports on the cytotoxic properties of five alkaloids from different Haplophyllum. species on HeLa and HCT-116 cell lines (Jansen et al., 2006).
The presence of diverse classes of lignans and alkaloids in different species of the genus Haplophyllum. and reports on the cytotoxicity of some of these compounds such as patavine, justicidine (Innocenti et al., Citation2002), and skimmianine (Jansen et al., 2006) prompted us to investigate the in vitro. cytotoxic effects of four Haplophyllum. species growing in Iran on various tumor cell lines from different origins. These species included H. tuberculatum., H. acutifolium. (DC.) G. Don, H. stapfianum. Hand.-Mazz., and H. viridulum. Soják of which the last two species are endemic in Iran. In addition, Ruta graveolens. L., with known cytotoxic compounds (Pathak et al., Citation2003; Wu et al., Citation2003), which genus is taxonomically the closest one to the genus Haplophyllum. (Townsend, Citation1986), was evaluated for its cytotoxic effect as a comparison with Haplophyllum. species.
Materials and Methods
Plant material
The aerial parts of plants were collected from the Fars province of Iran in July 2003 and identified by Mohammad Soltani. Voucher specimens were deposited in the Herbarium of Pharmacognosy, Faculty of Pharmacy, Shiraz University of Medical Science.
Extraction of the plant material
Dried and powdered plant material (1.6 g) was dissolved in 16 mL of absolute ethanol and sonicated two times for 1 min at room temperature. Extraction was continued by the addition of 144 mL of ethanol (67%) and sonicated at 70°C for 10 min. The extract was concentrated under reduced pressure by rotary evaporation below 45°C and then lyophilized to yield dried powder. The yield of dried extract as percentage weight of starting dried plant material for each plant species was as follows: H. tuberculatum., 23.1%; H. acutifolium., 21.9%; H. stapfianum., 20.1%; H. viridulum., 26.1%; and R. graveolens., 24.2%.
Preparation of human peripheral blood mononuclear cells and cell lines
Mononuclear cells were isolated from healthy volunteers by Ficoll (Amersham Bioscience, Amersham, UK) gradient centrifugation. Mononuclear cells were washed three times with RPMI 1640 (Gibco, Eggenstein, Germany) and then resuspended in the same medium supplemented with 10% heat-inactivated fetal calf serum (Gibco) at a concentration of 1 × 106 cells/mL. Cells were activated with 10 µg/mL phytohemagglutinin (PHA; Baharestan, Iran) and cultured in 96-well microplates (Nunc, Roskilde, Denmark).
The list of hematopoietic and nonhematopoietic cell lines used in this study is provided in . Adherent and non-adherent cell lines were seeded in 50 µL plastic culture flasks (Nunc) in RPMI 1640 or DMEM media (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 µL/mL streptomycin, 100 IU/mL penicillin, and incubated at 37°C in a 5% CO2 incubator.
Table 1. List of cell lines used in the cytotoxicity assay.
WST-1 cytotoxicity assay
The assay was based on the cleavage of the tetrazolium salt WST-1 (Roche, Mannheim, Germany) producing a soluble formazan salt; 96-well tissue culture microplates (Nunc) were seeded with 90 µL medium containing cells in suspension at proper cell counts, which was 20,000 cells/well for MCF-7, HeLa, and A549, 30,000 cells/well for all hematopoietic cell lines, SK-OV-3, and U937, and 40,000 cells/well for MDA-MB-453 and LNCap-FGC-10. After at least 6 h incubation, cells were treated with 10 µL of different concentrations of the extracts, positive controls, and solvent controls. Different concentrations of the extracts (by preparing twofold serial dilutions from an original 5 mg/mL stock solution), positive controls (doxorubicin, etoposide, and vinblastine), and solvent controls (PBS/ethanol) were added to the cells in 10 µL volumes in triplicate. The final concentration of extracts in each well was 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 µL/mL. Three wells containing only the same number of the cells were left as negative controls in each plate. Different concentrations of the solvent were tested for their probable cytotoxic effect. After 48 h incubation in a CO2 incubator (37°C), 10 µL of WST-1 was added to the test wells and the plate was further incubated at 37°C for 4 h. The absorbance was then determined at 450 nm and a reference wavelength of 630 nm by an ELISA spectrophotometer (Anthos 2020, Salzburg, Austria).
Statistical analysis
The statistical significance of the data was determined by one-way ANOVA test using SPSS software version 10. p values less than 0.05 were taken as significant.
Results and Discussion
The cytotoxicity of the sonicated extract of plants on different cell lines and mononuclear cells was assessed using the WST-1 assay. All results were expressed as IC50 values, the concentration of the extract that inhibited cell proliferation up to 50% of the negative control. Cytotoxicity curves were obtained by plotting percentage of viability against different concentrations of the extract. Viability percent was calculated from the following equation:
The IC50 values were mathematically interpolated and are shown in .
Table 2. IC50 values for Haplophyllum. and Ruta. extracts tested against tumor cell lines and PBMCs.
No major solvent cytotoxicity was observed on experimented cell lines (data are not shown).
In order to compare the cytotoxic effect of extracts on different cell lines, three groups were considered according to the concentrations of extracts that were used in this study: IC50 less than 31.3 µg/mL (high effect), IC50 between 31.3 and 62.5 µg/mL (moderate effect), and IC50 higher than 62.5 µg/mL (low effect).
According to the classification mentioned above, high effect of H. tuberculatum. extract was observed on RAMOS, U937, and RPMI-8866 cells, which was significantly more than the effect on Jurkat cell line (p < 0.05), and among solid tumors it had a high cytotoxic effect on LNCap-FGC-10 and 5637. H. stapfianum. showed high cytotoxicity on RAMOS and U937, which was significantly more than the two other hematopoietic cell lines (p < 0.05). Among solid tumors, a high effect of this species was observed against LNCap-FGC-10 and a moderate effect against 5637 cell line. H. acutifolium. showed high cytotoxicity against RAMOS and moderate effect against U937 cell line. This species had a high effect on LNCap-FGC-10 and moderate effect on 5637 cell line. We observed a low effect of H. viridulum. on all tumor cell lines except for RAMOS on which it showed moderate cytotoxicity. In a comparison between IC50 values obtained from investigated Haplophyllum. species (except for H. viridulum.) and R. graveolens., lower IC50 values were achieved against all tumor cell lines except for LNCap-FGC-10.
We also investigated the cytotoxic effect of three Haplophyllum. species including H. tuberculatum., H. stapfianum., and H. acutifolium. on normal peripheral blood mononuclear cells (PBMCs) in order to see the adverse effect of the extract on these normal cells.
IC50 values for normal PBMCs and comparison with those of hematopoietic cell lines revealed that the cytotoxicity of H. stapfianum. extract on normal PBMCs was significantly less than the effect on two hematopoietic cell lines, RAMOS and U937 (p < 0.05). A lower cytotoxic activity of H. acutifolium. was also observed on normal PBMCs than on RAMOS.
In conclusion, our data suggest that H. stapfianum. as an endemic species is a good candidate for fractionation and purification to find its active putative compound(s). The significant cytotoxic effects on both solid and hematopoietic cell lines and respectively lower cytotoxicity on PBMCs gives promise on finding selective anticancer agent(s). H. tuberculatum. and H. acutifolium. can also be at the second line of investigation.
Acknowledgment
This work was financially supported by a grant from Shiraz Institute for Cancer Research (grant no. ICR-82-103). The authors are grateful to Dr. A. R. Khosraui, Director of the Herbarium of Shiraz University, for his valuable help.
References
- Evcim U, Gozler B, Freyer AJ, Shamma M (1986): Haplomyrtin and (-)-haplomyrfolin, two new lignans from Haplophyllum myrtifolium.. Phytochemistry 25: 1949–1951.
- Gozler B, Önür MA, Gozler T, Kadan G, Hesse M (1994a): Lignans and lignan glycosides from Haplophyllum cappadocicum.. Phytochemistry 37: 1693–1698.
- Gozler B, Kivcak B, Arar G, Gozler T, Hesse M (1994b): (-)-Padocin: A novel epoxylignan from Haplophyllum cappadocicum.. Heterocycles 39: 243–249.
- Gozler B, Rentsch D, Gozler T, Unver N, Hesse M (1996): Lignans, alkaloids and coumarins from Haplophyllum vulcanicum.. Phytochemistry 42: 695–699.
- Graham JG, Quinn ML, Fabricant DS, Farnsworth NR (2000): Plants used against cancer, an extension of the work of Jonathan Hartwell. J Ethnopharmacol 73: 347–377.
- Innocenti G, Puricelli L, Piacente S, Caniato R, Filippini R, Cappelletti EM (2002): Patavine: A new arylnaphthalene lignan glycoside from shoot cultures of Haplophyllum patavinum.. Chem Pharm Bull 50: 844–846.
- Jansen O, Akhmedjanova V, Angenot L, Balansard G, Chariot A, Ollivier E, Tits M, Frederich M (2006): Screening of 14 alkaloids isolated from Haplophyllum. A. Juss. for their cytotoxic properties. J Ethnopharmacol 105: 241–245.
- Khalid SA, Farouk A, Geary TG, Jensen JB (1986): Potential antimalarial candidate from African plants: An in vitro. approach using Plasmodium falciparum.. J Ethnopharmacol 15: 201–209.
- Pathak S, Multani AS, Banerji P (2003): Ruta. 6 selectively induces cell death in brain cancer cells but proliferation in normal peripheral blood lymphocytes: A novel treatment for human brain cancer. Int J Oncol 23: 975–982.
- Puricelli L, Innocenti G, Delle Monache G, Caniato R, Filippini R, Cappelletti, EM (2002): In vivo. and in vitro. production of alkaloids by Haplophyllum patavinum.. Nat Prod Lett 16: 95–100.
- Sheriha GM, Abouamer K, Elshtaiwi BZ, Ashour AS, Abed FA, Alhallaq HH (1987): Quinoline alkaloids and cytotoxic lignans from Halophyllum tuberculatum.. Phytochemistry 26: 3339–3341.
- Townsend CC (1986): Taxonomic revision of the genus Haplophyllum. (Rutaceae). Hooker's Icones Plantarum, vol. XL, parts I, II & III. Kent, Bentham-Moxon Trustees, pp. 14, 16.
- Ulubelen A, Mericli AH, Mericli F, Tan N (1993): Two new coumarins from Haplophyllum ptilostylum.. J Nat Prod 56: 1184–1186.
- Wu TS, Shi LS, Wang JJ, Iou SC, Chang HC, Chen YP, Kuo YH, Chang YL, Teng CM (2003): Cytotoxic and antiplatelet aggregation principles of Ruta graveolens.. J Chin Chem Soc 50: 171–178.
- Yuldashev MP (2001): Flavonoids of Haplophyllum foliosum. and H. pedicellatum.. Chem Nat Comp 37: 288–289.
- Yuldeshev MP (2002): Flavonoids and coumarins of Haplophyllum leptomerum. and H. dubium.. Chem Nat Comp 38: 192–193.