Diverse Biological Activity of Odontioda Marie Noel ‘Velano’ Extracts

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).

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Figure 1.

Odontioda Marie Noel ‘Velano’.

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).

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Figure 2.

Sequential fractionation of MeOH with organic solvents.

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).

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Figure 3.

Effect of O. Marie Noel ‘Velano’ extracts on UV-induced cytotoxicity. HSC-2 cells were exposed to UV irradiation (6 J/m²/min, 1 min) in PBS(−) containing MeOH extract (A), n-hexane (B), EtOAc (C), n-BuOH (D) or H₂O (E) fraction of the bulb of O. Marie Noel ‘Velano’. After 48 hours' incubation in fresh medium, the viable cell number (expressed as a % of control cells, before final incubation that had not been exposed to UV irradiation) was determined. Mean±S.D. of triplicate assays.

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.