Abstract
Background: Several pharmacologically active substances have been isolated from orchid plants other than Odontioda Marie Noel ‘Velano’. Whether or not MeOH extract fractions from O. Marie Noel ‘Velano’ bulbs exert various biological activities was investigated. Materials and Methods: The MeOH extract was stepwise separated by organic solvents into n-hexane, EtOAc, n-BuOH and H2O layer fractions. Cytotoxic activity against human tumor and normal cells was determined by the MTT method. Nitric oxide (NO) was determined by the Griess method. Osteoclastogenesis was monitored by tartrate-resistant acid phosphatase (TRAP) activity. Result: The EtOAc fraction showed the highest tumor-specific cytotoxicity, followed by the n-hexane and other fractions. The EtOAc and n-BuOH fractions protected the cells from the cytotoxicity induced by UV irradiation. The EtOAc and n-hexane fractions inhibited NO production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells. The EtOAc fraction most strongly inhibited the receptor activator for nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, followed by the n-BuOH, n-hexane and H2O fractions. Conclusion: Most of the biological activities tested were concentrated in the EtOAc fraction, and separation from cytotoxic substances is needed to identify the active principle(s).
- Odontioda Marie Noel ‘Velano’ extracts
- cytotoxicity
- UV protection
- macrophage
- LPS
- RANKL
- NO
- osteoclastogenesis
Various pharmacologically active substances have been isolated from the stalks of some orchid species (Prosthechea michuacana, Dendrobium nobile, Bletilla striata, Dendrobium densiflorum, Cremastra appendiculata). These compounds have shown radical-scavenging, antioxidant (1), antifibrosis (2), antitumor (3), antimicrobial (3, 4), and antiplatelet aggregation (5) activities, growth modulation of mouse T and B lymphocytes (6) and antimuscarinic activity (7). However, compounds have not yet been isolated from another species of orchid, Odontioda Marie Noel ‘Velano’ (Figure 1). A study to isolate new compounds from this species was therefore initiated. As a first step, the MeOH extract of O. Marie Noel ‘Velano’ was roughly separated into four fractions by stepwise partitions with organic solvents, and whether or not these separated fractions showed various biological activities, such as tumor-specific cytotoxicity, protection of cells from the cytotoxicity induced by ultraviolet (UV) irradiation, inhibition of nitric oxide (NO) production by lipopolysaccharide (LPS)-activated macrophage-like cells and inhibition of receptor activator for nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis was investigated.
Materials and Methods
Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA); fetal bovine serum (FBS) (SAFC Biosciences, St. Louis, MO, USA); RPMI-1640 mediun, α-minimal essential medium (α-MEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS from Escherichia coli (serotype 0111:B4) (Sigma-Aldrich, St. Louis, MO, USA); dimethyl sulfoxide (DMSO) (Wako Pure Chemical, Osaka, Japan) and RANKL (R & D Systems, Minneapolis, MN, USA).
Sequential fractionation with organic solvents. Bulbs of O. Marie Noel ‘Velano’ (Orchidaceae) were supplied from Nichirei Garden, Nagano Prefecture, Japan, during the April of 2009. This specimen was proved and identified by Mr. H. Sumiyoshi (Nichirei Garden) and also a voucher specimen (#20090617) is deposited in the Medicinal Plant Garden of Josai University.
The air-dried bulbs of O. Marie Noel ‘Velano’ (63.6 g) were extracted three times with MeOH under reflux for 3 hours. H2O (80 ml) was added to the MeOH solution (800 ml) which was then partitioned with n-hexane (800, 400, 400 ml). The aqueous MeOH layer solution was evaporated, and H2O (100 ml) was added to the residual aqueous solution (100 ml) which was then extracted with EtOAc (200, 100, 100 ml) and n-BuOH (100, 50, 50 ml), successively. The n-hexane and EtOAc layer solutions were dried over anhydrous Na2SO4, and evaporated to dryness in vacuo to yield n-hexane (0.98 g) and EtOAc (0.94 g) fractions respectively. The n-BuOH and part of the aqueous layer solutions were also evaporated to give n-BuOH (1.02 g) and H2O fractions, respectively (Figure 2).
Cell culture. Human promyelocytic leukemia HL-60 cells were provided by Professor K. Nakaya, Showa University, Japan. Human oral squamous cell carcinoma cell lines (HSC-2, HSC-3, HSC-4) were provided by Professor M. Nagumo, Showa University. Normal human oral cells, gingival fibroblast (HGF), pulp cells (HPC) and periodontal ligament fibroblast (HPLF) were prepared from periodontal tissues, according to the guideline of the Intramural Ethic Committee (No. A0808), after obtaining informed consent from a 12-year old patient at the Meikai University Hospital. Since normal oral cells have a limited lifespan of 43-47 population doubling levels (PDL) (8), they were used at 8-15 PDL. Mouse macrophage-like RAW264.7 cells (9) were purchased from Dainippon Sumitomo Pharma, Osaka, Japan. The HL-60 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate under a humidified 5% CO2 atmosphere. The other cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. The normal cells were detached with 0.25% trypsin-0.025% EDTA-2Na in phosphate-buffered saline without Mg2+ and Ca2+ (PBS(−)) and subcultured at a 1:4 split ratio once a week, with a medium change in between the subcultures. The tumor cell lines were similarly trypsinized and subcultured.
Assay for cytotoxic activity. The cells (3×103 cells/well, 0.1 ml/well) were seeded in 96-microwell plates (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated for 48 hours to allow cell attachment. Near-confluent cells were treated for 48 hours with various concentrations of the test compounds in fresh medium. The relative viable cell number of adherent cells (except for HL-60 cells) was then determined by the MTT method. In brief, control and sample-treated cells were incubated for 4 hours with 0.2 mg/ml of MTT in the culture medium. After removing the medium, the reaction product, formazan, was extracted with DMSO and the absorbance (the relative viable cell number) was measured at 540 nm by a microplate reader (Multiskan Bichromatic Labsystems, Helsinki, Finland). The viability of the suspended cells, i.e. HL-60, was determined by cell counting with a hemocytometer after staining with 0.15% trypan blue. The 50% cytotoxic concentration (CC50) was determined from the dose–response curve. The tumor specificity index (TS) was calculated by the following equation: TS=mean CC50 (normal cells)/mean CC50 (all tumor cell lines).
Assay of UV protection. HSC-2 cells, that showed the highest sensitivity against UV irradiation among 6 adherent cell lines tested (8), were inoculated into 96-microwell plates (3×103 cells/well, 0.1 ml/well) and incubated for 48 hours to allow cell attachment. The culture supernatant was replaced with PBS(−) that contained various concentrations of the test substances, placed at 21 cm distance from a UV lamp (wavelength: 253.7 nm) and exposed to UV irradiation (6 J/m2/min) for 1 min. The PBS was replaced with fresh DMEM containing 10% FBS, and the cells were incubated for a further 48 hours to determine the relative viable cell number by MTT method, as described above.
Effect on NO production by macrophage-like cells. The RAW264.7 cells (6×104 cells/well, 0.1 ml/well) were inoculated into 96-microwell plates and incubated for 3 hours. The medium was then replaced with phenol red-free DMEM containing 10% FBS and the indicated concentrations of the test samples with or without LPS (100 ng/ml). LPS stimulates NO production via induction of inducible NO synthase (iNOS) expression (10). After incubation for 24 hours, the NO released into the culture supernatant was measured by the Griess method. From the dose–response curve, the 50% effective concentration (EC50) was calculated. After removing the medium, the attached cells were stained with MTT reagent to determine the CC50, as described above. The selectivity index (SI) for the inhibition of NO production was determined by the following equation: SI=CC50/EC50 (11).
Osteoclastogenesis in cell culture. The RAW264.7 cells were suspended in α-MEM supplemented with 5% FBS and seeded at 2×103 cells/well in 96-microwell plates (0.1 ml/well) in the presence or absence of RANKL (10 ng/ml) with or without the test samples. After 4 days in culture, tartrate-resistant acid phosphatase (TRAP) activity of the medium was determined and TRAP staining of the cells was performed.
Measurement of TRAP activity and TRAP-staining. The culture media (30 μl) were incubated for 30 min at 37°C with 30 μl of 600 mM sodium acetate buffer (pH 5.5) containing L-ascorbic acid (17.6 mg/ml), sodium tartrate dehydrate (9.2 mg/ml), disodium 4-nitrophenylphosphate (3.6 mg/ml), Triton X-100 (0.3%), EDTA (6 mM) and NaCl (600 mM). The reaction was terminated by the addition of 30 μl of NaOH (300 mM) and the absorbance at 405 nm was measured by microplate reader. TRAP histochemical staining of the cells was performed using a leukocyte acid phosphatase kit (Sigma-Aldrich). The cultured cells were fixed with 100% MeOH for 1 min at room temperature and air-dried, then stained for TRAP activity.
Statistical analysis. The difference between two groups was evaluated by Student's t-test.
Results
Cytotoxicity. The MeOH extract showed the highest cytotoxicity against the HL-60 cells (CC50=106 μM), followed by HSC-4 (CC50=188 μM) > HSC-3 (CC50≥221 μM) > HSC-2 (CC50≥246 μM) (Table I). The human normal oral cells (HGF, HPC, HPLF) showed lower sensitivity to the MeOH extract (CC50>239 μM), with TS of 1.3 (Table I).
Among the four fractions prepared from the MeOH extract (Figure 2), the EtOAc fraction showed the highest cytotoxicity, and the highest tumor-specificity TS 2.1, followed by the n-hexane (TS=1.5) > n-BuOH and H2O fractions (TS=1) (Table I). It should be noted that the order of sensitivity to the EtOAc fraction was in the same order as that to the MeOH extract: HL-60 (CC50=35 μM) > HSC-4 (CC50=45 μM) > HSC-3 (CC50=95 μM) > HSC-2 (CC50=106 μM) > normal cells (HGF, HPC, HPLF) (CC50=133-166 μM) (Table I).
UV protection activity. One minute exposure of the HSC-2 cells to UV irradiation induced irreversible cell death after 48 hours incubation (Figure 3). Addition of the EtOAc fraction to the irradiation buffer markedly protected the cells from the cytotoxicity induced by UV irradiation. The UV protection effect of the EtOAc fraction was increased with increasing concentrations up to 500 μg/ml, where some cytotoxicity was observed (Figure 3C). The n-BuOH fraction showed slightly lower UV protective activity (Figure 3D). The MeOH extract, n-hexane and H2O fractions (Figure 3A, B and E, respectively) displayed no UV-protective effects.
NO production by macrophage-like cells. Treatment of the RAW264.7 cells with LPS significantly elevated the extracellular concentration of NO from the background level to 14-15 μM (Figure 4). Simultaneous addition of the EtOAc fraction (Figure 4C) inhibited the LPS-stimulated NO production (EC50=8.9 μg/ml, CC50=90.6 μg/ml, SI=10.2). The n-hexane fraction (Figure 4B), which was much less cytotoxic (CC50=205.2 μg/ml), produced a comparable SI value (SI=10.7). On the other hand, crude MeOH extract (SI=5.0, Figure 4A), n-BuOH (SI=3.1, Figure 4D) and H2O (SI=1.0, Figure 4E) fractions showed much less or no inhibitory activity on the NO production (Table II).
Effect on osteoclastogenesis. Treatment of the RAW264.7 cells with RANKL stimulated the production of osteoclasts (Figure 5A), whereas the MeOH extract and its fractions except for the n-BuOH fraction were inactive in osteoclastogenesis (Figure 5B, upper panel). On the other hand, the MeOH extract and its fractions significantly (p<0.01) inhibited the RANKL-stimulated osteoclastogenesis at higher concentrations (16 or 80 μg/ml) (Figure 5B, lower panel). Among them, the EtOAc fraction showed the most potent inhibitory effect, followed by the n-BuOH fraction > n-hexane fraction > MeOH extract >> H2O fraction.
Discussion
The present study demonstrated, for the first time, several biological activities of the MeOH extracts of the bulb of O. Marie Noel ‘Velano’. Among the four different fractions of the MeOH extract, the EtOAc fraction showed several interesting biological activities: higher cytotoxicity against human tumor cells (three oral squamous cell carcinomas and one promyelocytic leukemia) than against human normal oral cells, with a TS value of 2.1 (Table I); protection of the cells from UV-induced damage (Figure 3); inhibition of NO production by LPS-activated RAW264.7 cells (Figure 4, Table II) and RANKL-stimulated osteoclastogenesis (Figure 5), suggesting its possible anti-inflammatory activity. However, the EtOAc fraction was found to be the most cytotoxic (Table I). We have recently separated the EtOAc fraction by silica gel column chromatography into a flow through fraction and adsorbed fractions which were then eluted stepwise, in order to separate the active principle(s) from the cytotoxic substance(s). The biological activities of these fractions will be reported elsewhere.
The present study also demonstrated that the n-BuOH fraction also showed minor stimulation of osteoclastogenesis although this fraction inhibited the RANKL-induced osteoclastogenesis, suggesting that the n-BuOH fraction contains both stimulator and inhibitor of osteoclastogenesis. Further fractionation is necessary to separate these substances.
Acknowledgements
We are grateful to Nichirei Garden Corporation, Japan, for their kind supply of O. Marie Noel ‘Velano’.
- Received November 29, 2010.
- Revision received February 2, 2011.
- Accepted February 3, 2011.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved